Immune landscape in children with X-linked retinoschisis

Immune landscape in children with X-linked retinoschisis | 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 Immune landscape in children with X-linked retinoschisis Ying Hsu, Giulia Del Valle, Sarah T. Stanley, Brianna Lobeck, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6263927/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background. X-linked retinoschisis is a retinovitreal disorder primarily affecting males, starting in childhood. Over time, patients experience deterioration of vision due to the lack of retinoschisin-1 function. In clinical trials performing ocular gene delivery in those affected by this disorder, ocular inflammation was observed, which may have masked efficacy. A subsequent study focusing on analyzing the populations of peripheral blood mononuclear cells and cytokines in adults with this disease reported aberrant dendritic cell numbers and cytokine levels in peripheral blood. Adults with this disease may have an altered baseline immunity. However, children with X-linked retinoschisis were not included in that study, and whether the aberrant peripheral immunity in affected adults was a consequence of advanced eye pathology remained unclear. This study focuses on analyzing the populations of blood lymphocyte subsets in children aged 0 to 7 with X-linked retinoschisis and age-matched controls using flow cytometry. Results. The fractions of lymphocyte subsets that were CD16a + were significantly lower in children with X-linked retinoschisis. The fractions of lymphocyte subsets that were CD16a+/CD56+, namely natural killer cells, were also significantly lower. In children with X-linked retinoschisis, the fractions of CD3+/CD4 + T cells were higher, and the fractions of CD3 + CD8 + T cells were lower, despite having the same amounts of total CD3 + T cells within their lymphocyte populations. This resulted in a significantly greater CD4/CD8 ratio in children with X-linked retinoschisis compared to age-matched controls. Conclusions. Alterations were found in blood lymphocyte compositions of children with X-linked retinoschisis within both innate and adaptive immune axes. Some alterations including an elevation of CD4/CD8 ratio in X-linked retinoschisis mirror those previously found in adult patients with this disease. The fact that these abnormalities were present early in this disease indicates that retinoschisin-1 may play a role in regulating immunity in addition to retinal adhesion. The findings may have implications for future treatments such as ocular gene delivery. X-linked retinoschisis retinoschisin flow cytometry CD4/CD8 ratio. Figures Figure 1 Figure 2 Figure 3 Background X-linked retinoschisis (XLRS) is a retinovitreal disorder that causes visual impairment primarily in males. This disease is characterized by formation of fluid-filled cysts separating retinal layers, a clinical feature referred to as retinoschisis that begins at a young age. XLRS is caused by mutations in the retinoschisin-1 ( RS1 ) gene, located on the X chromosome. RS1 encodes for the protein RS1 important for retinal adhesion ( 1 ). The median age at onset of symptoms for patients with XLRS is 4 years (but can start as young as 1 year old), and the median age at diagnosis is 7 years ( 2 ). By 12 years old, patients with this disease usually have mild visual impairment ( 2 ), and would typically possess low vision by the time they are about 25 years old, since visual acuity declines annually ( 2 ). Approximately 13% of patients experience vitreous hemorrhage at a median age of 9.4 years and about 9% experience retinal detachment at a similar age, which contributes to temporary or permanent vision loss in this disease ( 2 ). Gene therapy treatments in animal models with XLRS show promise for treating this disease using adeno-associated viruses (AAVs) as delivery vehicles ( 3 – 6 ). However, two phase I/II clinical trials testing gene augmentation therapies in XLRS patients did not show significant benefit. In one trial, the intravitreal injection of an AAV8 gene delivery vector containing the RS1 gene caused ocular inflammation in adult patients with XLRS treated with the high dose (1×10 11 − 3×10 11 vector genomes per eye, vg/eye) ( 7 ). Antibodies against the AAV8 capsid were observed, the levels of which appeared to be related to the severity of side effects ( 7 ). In another open label, phase I/II dose escalation clinical trial using the AAV2tYF-CB- RS1 gene therapy vector to treat XLRS, varying degrees of intraocular inflammation were observed, including blurred vision, cells in anterior chamber, vitreous cells or vitritis, most of which were successfully treated with steroids. Three out of 13 (23%) of the participants in the high dose group (who received 6 × 10 11 vg/eye) developed chronic uveitis, an ocular inflammatory condition that required treatment with steroids ( 8 ). These observations prompt the question whether certain subjects experience more severe side effects due to their baseline immunity, since uveitis is a condition that involves activation of T and B cells (reviewed in ( 9 )). In study participants, the duration of topical steroid administration appeared to be associated with the titers of AAV neutralizing antibodies in serum ( 8 ). Varying levels of neutralizing antibody in response to gene therapy may point to differences in B cell reactivity among the participants. These inflammatory conditions may have masked efficacy in these trials. To determine the immune baseline of XLRS patients and identify any abnormality that may interfere with gene therapy administration, a follow-up study was performed in adults with this disease using flow cytometry. Flow cytometry can be used to identify and quantify populations of immune cells by labeling cells in blood or tissues with cell type specific markers. Identification of circulating dendritic cells and their subtypes was of particular interest, since classical myeloid dendritic cells are professional antigen presenters that trigger the adaptive immune response, and the unconventional plasmacytoid dendritic cells are adept at secreting cytokines in response to viral pathogens (reviewed in ( 10 )). In the presence of pathogens, myeloid dendritic cells migrate to the lymph nodes to present the antigens to naïve T cells. The populations of CD11c + myeloid dendritic cells in the peripheral blood of adults with XLRS were significantly smaller than those in age-matched control subjects, whereas their populations of CD123 + plasmacytoid dendritic cells were elevated ( 11 ). Additionally, abnormal cytokine profiles were noted at baseline including elevated serum interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF)-α levels ( 11 ). These findings imply that the baseline peripheral immunity of patients with XLRS may be altered, but the functional implications of these differences are unclear. The XLRS subjects included in that study ranged from 23 to 72 years of age ( 11 ), and the immunity of children with XLRS was not investigated. Therefore, it is unclear if these differences in immune cell populations identified by flow cytometry were caused by worsening ocular pathologies in adult subjects during disease progression. A clinical study was initiated to investigate the baseline immune status of patients with XLRS from 0–100 years, for which the recruitment is onging. In this current report, initial flow cytometry findings from the cohort aged 0–7 including five children with XLRS and five age-matched control subjects are presented. Methods Patient recruitment The research was conducted in accordance with IRB #201911037 approved by the Institutional Review Board at the University of Iowa. Patients with molecularly confirmed XLRS were identified through the pediatric inherited eye disorder clinic. They were offered enrollment in the study and if accepted, asked to give a blood sample. Informed consent was obtained from the parents or guardian. All recruited patients were males due to the X-linked nature of this disease. Age-matched male control subjects without the diagnosis of a genetic eye disease were recruited from the general pediatric ophthalmology clinic; some control subjects underwent standard of care related surgical procedures and had the option for the blood sample to be obtained while under sedation. The blood samples obtained from XLRS patients and control subjects were analyzed by the University of Iowa Hospitals and Clinics Pathology Core Laboratory. The tests performed were complete blood counts with differentials and flow cytometry. For complete blood counts with differentials, whole blood was labeled with specific markers and visualized by XN-20 (Sysmex Corporation). Differentials were performed on a Sysmex SP50 slide stainer and imager using an automated software, Cellavision (Sysmex Corporation). For flow cytometry, peripheral blood mononuclear cells (PBMCs) were isolated via density gradient centrifugation. Antibody labeling and washes were performed following standard procedures established and validated in the Pathology Core Laboratory at the University of Iowa. A 10-color FACSCanto flow cytometer (BD Biosciences) was used and results were analyzed on FlowJo (BD Biosciences) or CytoPaint Classic immunophenotyping software (Leukobyte). Standard flow cytometry panel plus the CD16 and Dendrite Cell Determination analyses were conducted. Data analysis Statistical analysis was performed using GraphPad PRISM. Comparison between groups were performed using unpaired t -test. Antibodies Antibodies used for flow cytometry are as follows: CD4-FITC (BD Biosciences, #340133), CD25-PE (BD Biosciences, #341010), CD3 PERCYPCY5.5 (BD Biosciences, #340948), CD16-PE CY7 (BD Biosciences, #335806), CD19-APC (BD Biosciences, #340722), CD56-APC R700 (BD Biosciences, #657887), CD8-APC H7 (BD Biosciences, #641409), CD127-BV421 (BD Biosciences, # 562436), CD45-V500 (BD Biosciences, # 647450), CD14-BV605 (BD Biosciences, #564054), CD11C-APC (BD Biosciences, #340544). Results Patient characteristics Subjects included in this report are a part of a larger, ongoing study with a target recruitment of 20 subjects with XLRS and 20 controls across different ages. In this report, findings from the youngest cohort including five children with XLRS aged 0-7 years and five age-matched control subjects are presented. The characteristics of these children with XLRS and age-matched controls, including genetic mutations and animo acid changes in those with XLRS, are listed in Table 1. The average ages between the children with and without XLRS are not significantly different (p = 0.7707, unpaired t-test). All recruited subjects are males due to the X-linked nature of this disease. Blood cell counts To determine if there are major differences in the populations of blood cells, complete blood counts with differentials were performed. There are no significant differences in absolute blood counts nor percentages of neutrophils, lymphocytes, monocytes, eosinophils, and basophils between children with XLRS and age-matched controls (Table 2). Flow cytometric analysis using markers associated with innate immunity To investigate populations of lymphocytes in PBMCs, whole blood was collected, PBMCs were isolated and stained with antibodies, and flow cytometry was performed. To enrich lymphocytes in subsequent analysis, flow cytometry data was gated by side scatter and CD45, a marker for cells of the hematopoietic lineage. This gating strategy, hereafter referred to as the lymphocyte gate (Figure 1a), excludes neutrophils, monocytes and granulocytes from subsequent analysis. To identify lymphocyte subsets related to innate immunity, cell populations were further gated by CD11c, CD16a, and CD56. CD11c is an integrin that plays a critical role in the adhesion of immune cells to endothelial cells prior to extravasation and immune activation. The population of CD11c+ cells includes monocytes, granulocytes, a subset of B cells, dendritic cells, and macrophages. Within the lymphocyte gate, children with XLRS tended to have lower percentages of cells that are CD11c+ (Figure 1b, Table 3). The trend of decrease in the population of CD11c+ cells in children with XLRS mirrors findings made in adult XLRS patients (11). The population of CD11c+ cells were then subdivided and gated by their expression of CD56. The percentage of CD11c+/CD56- cells in children with XLRS was not significantly different compared to that in age-matched controls (p = 0.6128, Figure 1c, Table 3). On the other hand, CD11c+/CD56+ cells are natural killer-like and are important for expansion of ɣδ T cells (12). The fractions of lymphocytes that were CD11c+/CD56+ was 40 % lower in children with XLRS compared to that in controls, but the difference between the groups did not reach statistical significance (p = 0.2200, Figure 1d, Table 3), likely due to the sample size of our study. Found on natural killer cells, a subset of monocytes, macrophages, and dendritic cells, CD16a is key to antibody-mediated cytotoxicity (13) as it binds to IgG with a 10-fold higher affinity compared to CD16b (14). The fraction of lymphocytes that are CD16a+ was significantly lower in blood samples collected from children with XLRS (p = 0.0388, Figure 1e, Table 3). On average, the percentage of CD16a+ cells in blood samples collected from children with XLRS is 66 % lower compared to that in age-matched controls. To further identify populations of natural killer cells, double positive for CD16a and CD56, populations were gated by these markers. About 10 % of lymphocytes in controls are CD16a+/CD56+, compared to only about 3 % in children with XLRS, and the difference between the groups was significant representing a significant (p = 0.0454, Figure 1f, table 3). In children with XLRS, populations of natural killer cells were 67 % lower compared to age-matched controls. Cell populations within the lymphocyte gate from a 3-year-old XLRS child (R4) and an age-matched control without XLRS (C3) are shown (Figure 1g). Remarkably, children with XLRS have fewer natural killer cells in their circulation. Flow cytometric analysis using markers associated with adaptive immunity To quantify lymphocyte subsets belonging to the adaptive immune system, cell populations in the lymphocyte gate were further gated by cell-specific markers (Figure 2a, Figure 2b). B cell counts, which were identified by CD19 expression, showed a trend of elevation in blood samples from children with XLRS, in which the counts were 41 % higher on average (p = 0.2348, Figure 2c, Table 3). Identified by CD3, T-cells play an important role in defenses against infections. T cells are further subdivided into CD4+ and CD8+ subsets, which are considered helper T cells and cytotoxic T cells, respectively. Of note, neither the fractions nor the absolute numbers of all CD3+ cells within the lymphocyte gate were different between children with and without XLRS (p = 0.9309 for fractions; p = 0.6968 for counts, Figure 2d, Table 3). However, the fraction of CD3+/CD4+ cells, that is the percentage of T cells that are helper T cells, is significantly higher in children with XLRS (p = 0.0333, Figure 2e, Table 3). Representative data is shown in Figure 2f. On the other hand, the fractions of CD3+/CD8+ cells, considered cytotoxic T-cells, are significantly lower in children with XLRS (p=0.0082, Figure 2g, representative data in Figure 2h, Table 3). The concomitant elevation in CD3+/CD4+ T-cells by 26 % and reduction in CD3+/CD8+ T cells by 29 % results in a higher CD4/CD8 ratio in children with XLRS (controls: 1.274 ± 0.125; XLRS: 2.234 ± 0.081, p = 0.0002). The elevation of CD4/CD8 ratio in children with XLRS mirrors the findings made in adult XLRS subjects by Mishra et al. (11). The populations of CD3+/CD4-/CD8- cells are not different between controls and children with XLRS (Figure 3a, Table 3). Within the CD4+ subset, T regulatory cells (Tregs) help modulate the immune response and immunosuppression. To determine whether the populations of Tregs are altered in XLRS, CD4+/CD25+/CD127− T cells were analyzed. T cells identified by CD4+/CD25+/CD127low or CD127− are analogous to classical Tregs identified by CD4+/CD25+/Foxp3+ (15). Foxp3 is the master transcriptional regulator for Treg-specific genes (16). Although not statistically significant, both the fractions and the absolute counts of CD4+/CD25+/CD127- cells within the lymphocyte gate show a trend of elevation in children with XLRS (Figure 3b, Figure 3c, Table 3). Populations of CD4+/CD25+/CD127- cells in blood are also non-significantly elevated in children with XLRS (Figure 3d, Table 3). Together, the data suggests that T cells shift toward a CD4 identify in children with XLRS. As a result of possessing a greater percentage of T cells that are CD4+ and a smaller percentage of T cells that are CD8+, children with XLRS possess a higher CD4/CD8 ratio, mirroring findings in adults with XLRS observed by Mishra et al. (11). The total numbers of T cells or lymphocytes were not different. A trend towards having a greater B cell pool is noted in children XLRS. Together, these findings suggest that children with XLRS also have an altered peripheral immunity, similar to observations in made adults. Discussion Apparent reductions in CD16a + and CD11c + cell populations in children with XLRS A previous study by Mishra et al. investigated the baseline immune status in adults with XLRS ( 11 ); they found aberrations in levels of CD11c + cells and an elevated CD4/CD8 ratio in adult subjects with XLRS ( 11 ). However, it was unclear whether these abnormalities in their peripheral immune cell populations developed over time as their ocular pathology progressed, or whether these alterations were present early. To address this, we have initiated a similar study in XLRS including subjects from different age groups. In this current study, initial findings in the youngest cohort of patients consisting of five children with XLRS and five age-matched controls aged between 0–7 are reported. In terms of markers commonly used to identify innate immune cells, significant reductions in populations CD16a + cells and CD16a+/CD56 + cells were identified. Notably, populations of CD16a + cells were reduced by 66% in children with XLRS, and populations of CD16a+/CD56 + natural killer cells were reduced by 67% in children with XLRS compared to controls. The reduction in this population may have functional consequences that merits further investigation. The CD16b isoform, on the other hand, identifies neutrophils ( 17 ) and granulocytes ( 18 ). Antibodies against CD16a can successfully distinguish natural killer cells from neutrophils ( 19 ). Presumably representing cells capable of responding to antibody-coated immune complexes, the functional consequence of this finding is unknown without further study. In our study, children with XLRS demonstrate a trend of having fewer CD11c+, CD11c+/CD56-, and CD111c+/CD56 + cells in their lymphocyte fractions. However, these changes were not statistically significant, likely due to the sample size in our study. A reduction in the dendritic cell population could mean impaired antigen presentation and/or cytokine secretion. A shift towards CD4 + T cell identity and elevation of CD4/CD8 ratio in children with XLRS Previously, an elevation of CD4/CD8 ratio was reported in adult subjects with XLRS at baseline when compared to that in age-matched controls (XLRS base-line: 3.5 ± 0.9; control: 2.2 ± 0.7; p < 0.001) ( 11 ). This finding opened the line of investigation whether an altered adaptive immunity could have impacted gene delivery in XLRS ( 11 ). Similarly, we found that children with XLRS also had a higher number of CD4 + T cells and a lower number of CD8 + T cells, resulting in an elevated CD4/CD8 ratio compared to age-matched control subjects. The fact that this abnormality is present early in XLRS suggests that it could be a primary feature of the disease, and not a consequence of advanced ocular pathology. CD4/CD8 ratio signals the balance between CD4 helper T cells and CD8 cytotoxic T cells. It is a prognostic marker in many diseases, including human immunodeficiency virus (HIV)-associated immune deficiency. In HIV, there is typically an inversion (decrease) in the CD4/CD8 ratio due to the rapid depletion of CD4 + T cells, signaling the loss of ability to fight opportunistic infections ( 20 ). Conversely, an increase in CD4/CD8 ratio may signal an increased immune competence. In an observational, multi-center, and retrospective study investigating factors contributing to COVID-19 disease severity, patients in the critically-ill group had higher CD4/CD8 ratios compared to those in the non-critically ill group after coronavirus infections ( 21 ). Furthermore, medications including tocilizumab and corticosteroids (methyl-prednisolone or dexamethasone) were more frequently used in the critically-ill group than in the non-critically-ill group (46% vs 4%, p = 0.0062 and 85% vs 36%, p = 0.0122, respectively). Taken together, these studies support the notion that possessing an elevated CD4/CD8 ratio, as is the case in children with XLRS, could predispose the subject to experiencing more severe inflammation when faced with viral infections or when undergoing treatments utilizing viral vectors. These findings could have implications for gene therapy development for this disease, such as the need for additional immunosuppressive treatments. However, these conjectures need to be further tested. In the context of viral vector-based gene delivery, the functional consequences of these immune alterations in XLRS are not known without further study. Changes in populations of peripheral immune cells may imply dysfunction, but not necessarily so. However, our report provides additional evidence that both the innate and adaptive immune systems are abnormal in children with XLRS. In the clinical gene therapy trial, elevation in AAV8 capsid-specific antibodies were observed in adults with XLRS after AAV8-mediated gene therapy, and the antibody levels were not the same among individuals receiving the same dose ( 7 ). In another clinical trial for treating XLRS, 23% of the study participants in the high dose group developed chronic uveitis ( 8 ), a condition related to activation of T and B cells (reviewed in ( 9 , 22 )), but others in the high dose group did not develop this condition. In our study, children with XLRS showed a trend of having greater populations of B cells in their blood, but the statistical power was limited by the sample size of our study. Determining how immune baseline measurements relate to outcomes would be of particular interest. Interestingly, RS1 expression is a prognostic marker for lung adenocarcinoma, and RS1 gene expression was lower in tumors ( 23 ). A previously unknown role of the RS1 gene in immunity may be emerging. Conclusions Alterations in immune cell populations belonging to both the innate and adaptive axes were found in peripheral blood collected from children with XLRS compared to age-matched controls. Some of these findings mirror observations made in adults with XLRS. Together, this supports the notion that aberrations in the peripheral immune compartment are a direct consequence of loss of RS1 function and not secondary to the worsening ocular pathologies in this disease. However, to demonstrate that this is a perturbation present since birth, this conclusion would be further supported by samples from newborn children with XLRS, or longitudinal studies. Declarations Ethics approval and consent to participate The research was conducted in accordance with Declaration of Helsinki and with IRB #201911037 approved by the Institutional Review Board at the University of Iowa. Informed consent was obtained from the parents or guardian. Clinical trial number: not applicable. Consent for publication This manuscript does not contain individually identifiable information. Availability of data and materials All data generated or analyzed during this study are included in this published article. Competing interests Authors do not have competing interests. Funding The study was supported by Chakraborty family fund (PI: Drack), and Knights Templar Eye Foundation career starter grant (PI: Hsu). The funders do not have a role in experimental design or data analysis. Authors' contributions AVD conceptualized the study. GDV, STS, CS, AD and AVD recruited and consented patients. SIS, YH and GDV interpreted the flow cytometry results. YH also analyzed the data, generated the figures and wrote the manuscript. BL, GDV and CRF assisted in data interpretation and scientific visualization. YH, BL, GDV, STS, CS, SS, CRF, AD and AVD critically revised the manuscript. Acknowledgements The authors would like to acknowledge funding sources including the Chakraborty Family Fund (PI: Drack), Knights Templar Eye Foundation Career Starter grant (PI: Hsu), and Ronald Keech Professorship. References Wu WW, Wong JP, Kast J, Molday RS. RS1, a discoidin domain-containing retinal cell adhesion protein associated with X-linked retinoschisis, exists as a novel disulfide-linked octamer. J Biol Chem. 2005;280(11):10721-30. Hahn LC, van Schooneveld MJ, Wesseling NL, Florijn RJ, Ten Brink JB, Lissenberg-Witte BI, et al. X-Linked Retinoschisis: Novel Clinical Observations and Genetic Spectrum in 340 Patients. Ophthalmology. 2022;129(2):191-202. Ou JX, Vijayasarathy C, Ziccardi L, Chen S, Zeng Y, Marangoni D, et al. Synaptic pathology and therapeutic repair in adult retinoschisis mouse by AAV-transfer. J Clin Invest. 2015;125(7):2891-903. Zeng Y, Takada Y, Kjellstrom S, Hiriyanna K, Tanikawa A, Wawrousek E, et al. RS-1 gene delivery to an adult Rs1h knockout mouse model restores ERG b-wave with reversal of the electronegative waveform of X-linked retinoschisis. Invest Ophthalmol Vis Sci. 2004;45(9):3279-85. Vijayasarathy C, Zeng Y, Marangoni D, Dong LJ, Pan ZH, Simpson EM, et al. Targeted Expression of Retinoschisin by Retinal Bipolar Cells in XLRS Promotes Resolution of Retinoschisis Cysts Sans RS1 From Photoreceptors. Invest Ophthalmol Vis Sci. 2022;63(11). Hassan S, Hsu Y, Thompson JM, Kalmanek E, VandeLune JA, Stanley S, et al. The dose-response relationship of subretinal gene therapy with rAAV2tYF-CB-hRS1 in a mouse model of X-linked retinoschisis. Front Med (Lausanne). 2024;11:1304819. Cukras C, Wiley HE, Jeffrey BG, Sen HN, Turriff A, Zeng Y, et al. Retinal AAV8-RS1 Gene Therapy for X-Linked Retinoschisis: Initial Findings from a Phase I/IIa Trial by Intravitreal Delivery. Mol Ther. 2018;26(9):2282-94. Pennesi ME, Yang P, Birch DG, Weng CY, Moore AT, Iannaccone A, et al. Intravitreal Delivery of rAAV2tYF-CB-hRS1 Vector for Gene Augmentation Therapy in Patients with X-Linked Retinoschisis: 1-Year Clinical Results. Ophthalmol Retina. 2022;6(12):1130-44. Zhu L, Chen B, Su W. A Review of the Various Roles and Participation Levels of B-Cells in Non-Infectious Uveitis. Front Immunol. 2021;12:676046. Fitzgerald-Bocarsly P, Dai JH, Singh S. Plasmacytoid dendritic cells and type I IFN: 50 years of convergent history. Cytokine Growth Factor Rev. 2008;19(1):3-19. Mishra A, Vijayasarathy C, Cukras CA, Wiley HE, Sen HN, Zeng Y, et al. Immune function in X-linked retinoschisis subjects in an AAV8-RS1 phase I/IIa gene therapy trial. Mol Ther. 2021;29(6):2030-40. Li W, Okuda A, Yamamoto H, Yamanishi K, Terada N, Yamanishi H, et al. Regulation of development of CD56 bright CD11c + NK-like cells with helper function by IL-18. PLoS One. 2013;8(12):e82586. Yeap WH, Wong KL, Shimasaki N, Teo EC, Quek JK, Yong HX, et al. CD16 is indispensable for antibody-dependent cellular cytotoxicity by human monocytes. Sci Rep. 2016;6:34310. Bruhns P, Iannascoli B, England P, Mancardi DA, Fernandez N, Jorieux S, et al. Specificity and affinity of human Fcγ receptors and their polymorphic variants for human IgG subclasses. Blood. 2009;113(16):3716-25. Yu N, Li X, Song W, Li D, Yu D, Zeng X, et al. CD4(+)CD25 (+)CD127 (low/-) T cells: a more specific Treg population in human peripheral blood. Inflammation. 2012;35(6):1773-80. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol. 2003;4(4):330-6. Victor AR, Weigel C, Scoville SD, Chan WK, Chatman K, Nemer MM, et al. Epigenetic and Posttranscriptional Regulation of CD16 Expression during Human NK Cell Development. J Immunol. 2018;200(2):565-72. Selvaraj P, Carpen O, Hibbs ML, Springer TA. Natural Killer Cell and Granulocyte Fc-Gamma Receptor Iii (Cd16) Differ in Membrane Anchor and Signal Transduction. J Immunol. 1989;143(10):3283-8. Liao B, Tumanut C, Li L, Corper A, Challa D, Chang A, et al. Identification of novel anti-CD16a antibody clones for the development of effective natural killer cell engagers. MAbs. 2024;16(1):2381261. Obeagu EI. Immunological Equilibrium: Unpacking CD4/CD8 Ratios in HIV. Infection. 2024;16:17. Pallotto C, Suardi LR, Esperti S, Tarquini R, Grifoni E, Meini S, et al. Increased CD4/CD8 ratio as a risk factor for critical illness in coronavirus disease 2019 (COVID-19): a retrospective multicentre study. Infect Dis (Lond). 2020;52(9):675-7. Smith JR, Stempel AJ, Bharadwaj A, Appukuttan B. Involvement of B cells in non-infectious uveitis. Clin Transl Immunology. 2016;5(2):e63. Zhang T, Cheng G, Chen P, Peng Y, Liu L, Li R, et al. RS1 gene is a novel prognostic biomarker for lung adenocarcinoma. Thorac Cancer. 2022;13(12):1850-61. Tables Table 1. Subject characteristics of children with XLRS and age-matched controls. Table 2. Leukocyte counts in peripheral blood in children with or without XLRS. Controls (absolute count) XLRS (absolute count) P value Neutrophils 41.040 ± 4.087 % (3054.000 ± 393.784 cells/µL) 41.500 ± 1.993 % (3212.000 ± 423.644 cells/µL) 0.9219 (0.7916) Lymphocytes 47.480 ± 4.698 % (3474.000 ± 325.032 cells/µL) 47.440 ± 2.946 % (3668.000 ± 489.504 cells/µL) 0.9944 (0.7498) Monocytes 8.040 ± 0.995 % (590.000 ± 71.972 cells/µL) 6.820 ± 0.801 % (514.000 ± 75.736 cells/µL) 0.3677 (0.4877) Eosinophils 2.640 ± 0.427 % (194.000 ± 31.559 cells/µL) 3.520 ± 1.059 % (242.000 ± 49.132 cells/µL) 0.4631 (0.4349) Basophils 0.580 ± 0.058 % (42.000 ± 3.742 cells/µL) 0.540 ± 0.154 % (38.000 ± 5.831 cells/µL) 0.8138 (0.5796) Table 3. Lymphocyte subsets in children with or without XLRS. Markers % lymphocyte gate in controls (absolute count) % lymphocyte gate in XLRS (absolute count) P value CD11c+ 11.158 ± 1.992 % (400.115 ± 101.864 cells/µL) 7.872 ± 1.035 % (274.099 ± 34.559 cells/µL) 0.1814 (0.2751) CD11c+/CD56+ 6.766 ± 1.956 % (240.404 ± 86.691 cells/µL) 4.058 ± 0.562 % (141.126 ± 19.633 cells/µL) 0.2200 (0.2965) CD11c+/CD56- 4.392 ± 0.724 % (159.710 ± 32.782 cells/µL) 3.814 ± 0.825 % (132.973 ± 28.941 cells/µL) 0.6128 (0.5579) CD16a+ 13.248 ± 3.299 % (467.071 ± 147.728 cells/µL) 4.520 ± 1.274 % (150.752 ± 43.493 cells/µL) 0.0388 (0.0740) CD16a+/CD56+ 10.318 ± 2.741 % (358.035 ± 118.271 cells/µL) 3.368 ± 1.050 % (112.628 ± 36.891 cells/µL) 0.0454 (0.0829) CD19+ 15.200 ± 2.223 % (544.000 ± 100.923 cells/µL) 20.800 ± 3.121 % (768.600 ± 142.742 cells/µL) 0.182 (0.2348) CD3+ 72.000 ± 3.332 % (2481.600 ± 219.593 cells/µL) 72.400 ± 2.977 % (2665.000 ± 397.288 cells/µL) 0.9309 (0.6968) CD3+/CD4+ 36.600 ± 3.140 % (1279.000 ± 176.070 cells/µL) 46.200 ± 2.035 % (1723.800 ± 276.160 cells/µL) 0.0333 (0.2115) CD3+/CD8+ 29.200 ± 2.035 % (996.400 ± 72.873 cells/µL) 20.800 ± 1.281 % (778.000 ± 132.950 cells/µL) 0.0082 (0.1877) CD3+/CD4-/CD8- 6.400 ± 1.122 % (212.000 ± 25.022 cells/µL) 5.400 ± 1.913 % (163.400 ± 21.860 cells/µL) 0.6641 (0.1817) CD3-/CD56+ 10.600 ± 2.731 % (369.800 ± 120.221 cells/µL) 5.000 ± 0.707 % (176.400 ± 28.151 cells/µL) 0.0824 (0.1559) CD4+/CD25+ 7.800 ± 0.735 % (264.200 ± 18.301 cells/µL) 9.200 ± 0.917 % (327.000 ± 44.714 cells/µL) 0.2675 (0.2299) CD4+/CD25+/CD127- 3.200 ± 0.374 % (108.000 ± 10.330 cells/µL) 4.200 ± 0.490 % (159.200 ± 30.871 cells/µL) 0.1434 (0.1544) Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 22 Apr, 2025 Reviews received at journal 22 Apr, 2025 Reviews received at journal 15 Apr, 2025 Reviewers agreed at journal 07 Apr, 2025 Reviewers agreed at journal 07 Apr, 2025 Reviewers invited by journal 28 Mar, 2025 Editor assigned by journal 28 Mar, 2025 Editor invited by journal 26 Mar, 2025 Submission checks completed at journal 25 Mar, 2025 First submitted to journal 25 Mar, 2025 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. 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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-6263927","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":443352909,"identity":"a5be23c5-e44d-4b0e-8619-c51c4359e114","order_by":0,"name":"Ying Hsu","email":"","orcid":"","institution":"University of Iowa","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Hsu","suffix":""},{"id":443352910,"identity":"655cea13-42d3-45a1-8485-6b64f6b8b1c3","order_by":1,"name":"Giulia Del Valle","email":"","orcid":"","institution":"University of Iowa","correspondingAuthor":false,"prefix":"","firstName":"Giulia","middleName":"Del","lastName":"Valle","suffix":""},{"id":443352911,"identity":"13681404-f018-4e6a-9c73-eb429328ec25","order_by":2,"name":"Sarah T. Stanley","email":"","orcid":"","institution":"University of Iowa","correspondingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"T.","lastName":"Stanley","suffix":""},{"id":443352912,"identity":"182c5e3e-1f29-44fa-8b3f-0dfba8de641d","order_by":3,"name":"Brianna Lobeck","email":"","orcid":"","institution":"University of Iowa","correspondingAuthor":false,"prefix":"","firstName":"Brianna","middleName":"","lastName":"Lobeck","suffix":""},{"id":443352913,"identity":"f75efcce-fc76-4ffa-b3e6-7506356a4460","order_by":4,"name":"Sergei I. Syrbu","email":"","orcid":"","institution":"University of Iowa Hospitals and Clinics","correspondingAuthor":false,"prefix":"","firstName":"Sergei","middleName":"I.","lastName":"Syrbu","suffix":""},{"id":443352914,"identity":"8f23ccb6-2ad6-4030-9451-0a3a8388c7f4","order_by":5,"name":"Christine Sinkey","email":"","orcid":"","institution":"University of Iowa","correspondingAuthor":false,"prefix":"","firstName":"Christine","middleName":"","lastName":"Sinkey","suffix":""},{"id":443352915,"identity":"4a50ad8c-dfbe-47a5-8ebf-c2fba27b7cf8","order_by":6,"name":"Christopher R. Fortenbach","email":"","orcid":"","institution":"University of Washington","correspondingAuthor":false,"prefix":"","firstName":"Christopher","middleName":"R.","lastName":"Fortenbach","suffix":""},{"id":443352916,"identity":"f1beef8b-6b17-49d4-acb2-36f05fa3c80b","order_by":7,"name":"Alina Dumitrescu","email":"","orcid":"","institution":"University of Iowa","correspondingAuthor":false,"prefix":"","firstName":"Alina","middleName":"","lastName":"Dumitrescu","suffix":""},{"id":443352917,"identity":"16545d23-aa20-434a-bc38-1bd8e8a24a05","order_by":8,"name":"Arlene V. Drack","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYFAC5gYQyQgkmRk+MEhABHnwamFEaGGcQbIWZrhKfFr4Zx9sfFxQwSC7vf3wY2PbNgs5g+MHGB+8bcOtReJcYrPxjDMMxnPOpBkn57ZJGBucSWA2nItHC8MZxjZp3rb/iTMYcpgPA7UkbjuQwAYUwa1D/gxj+2/efwyJM/jfMB+2BGk5/4D9Nz4tBkBbmHkbgFokcpiTGUFabiSwMePTYniGsVma5xiD8QyJZ8aGPeckjO1vPGyWnHMOtxa5M8wHP/PUMMjO4E9+LPGjrE5Osj/54Ic3ZXi8jwVAYmoUjIJRMApGAQUAAMDQTS7UAvhAAAAAAElFTkSuQmCC","orcid":"","institution":"University of Iowa Hospitals and Clinics","correspondingAuthor":true,"prefix":"","firstName":"Arlene","middleName":"V.","lastName":"Drack","suffix":""}],"badges":[],"createdAt":"2025-03-19 18:38:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6263927/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6263927/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80813646,"identity":"d2457250-4601-4c04-8002-0354712905c4","added_by":"auto","created_at":"2025-04-17 10:42:44","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":93540,"visible":true,"origin":"","legend":"\u003cp\u003eQuantification of lymphocyte subsets following flow cytometry using innate immune markers in children with X-linked retinoschisis (XLRS) and age-matched controls. Peripheral blood mononuclear cell populations were analyzed by flow cytometry in children with XLRS and controls. Following gating by side scatter and CD45 (a), cell populations within the lymphocyte gate that are CD11c+ (b), CD11c+/CD56- (c), CD11c+/CD56+ (d), CD16a+ (e), and CD16a+/CD56 (f) were quantified. Both fractions (%) and absolute counts (#) of each cell population are shown. Lymphocyte populations gated by CD16a and CD56 in a 3-year-old XLRS subject and an age-matched control are displayed (g).\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6263927/v1/6fef5f8678e97989960af920.png"},{"id":80813644,"identity":"dd354075-26b1-4aa5-8454-91c1a4acc15e","added_by":"auto","created_at":"2025-04-17 10:42:44","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":74766,"visible":true,"origin":"","legend":"\u003cp\u003eQuantification of lymphocyte subsets following flow cytometry using markers related to adaptive immunity in children with X-linked retinoschisis (XLRS) and age-matched controls. Peripheral blood mononuclear cell populations were analyzed by flow cytometry in children with XLRS and controls. Following gating by side scatter and CD45 (a), cell populations within the lymphocyte gate that are CD19+ (b, c), CD3+ (d), CD3+/CD4+ (e, f), CD3+/CD8+ (g, h) were quantified. Both fractions (%) and absolute counts (#) are shown. Lymphocyte populations for a 3-year-old control subject (C4) and a 3-year-old patient with XLRS (R3) gated by CD3 and CD4 (f) or CD3 and CD8 (h) are displayed.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6263927/v1/af992bdf9e2ea616dffcda82.png"},{"id":80813645,"identity":"20aa47d5-15dd-4dab-ad38-76c99b4fe729","added_by":"auto","created_at":"2025-04-17 10:42:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":49707,"visible":true,"origin":"","legend":"\u003cp\u003eQuantification of lymphocyte subsets following flow cytometry using markers related to adaptive immunity in children with X-linked retinoschisis (XLRS) and age-matched controls. Peripheral blood mononuclear cell populations were analyzed by flow cytometry in children with XLRS and controls. Following gating by side scatter and CD45, cell populations within the lymphocyte gate that are CD3+/CD4-/CD8- (a), CD4+/CD25+ (b, c), and CD4+/CD25+/CD127- (d) were quantified. Both fractions (%) and absolute counts (#) are displayed. Data for a 3-year-old control subject (C4) and a 3-year-old patient with XLRS (R3) is shown (c).\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6263927/v1/a9cc81edf3d5386ac873fbdf.png"},{"id":80815385,"identity":"acc6b0ef-1fb1-495d-81fa-7f5a592238e2","added_by":"auto","created_at":"2025-04-17 10:58:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":705077,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6263927/v1/eaa3c01d-54a6-4615-a829-3379473d5149.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Immune landscape in children with X-linked retinoschisis","fulltext":[{"header":"Background","content":"\u003cp\u003eX-linked retinoschisis (XLRS) is a retinovitreal disorder that causes visual impairment primarily in males. This disease is characterized by formation of fluid-filled cysts separating retinal layers, a clinical feature referred to as retinoschisis that begins at a young age. XLRS is caused by mutations in the retinoschisin-1 (\u003cem\u003eRS1\u003c/em\u003e) gene, located on the X chromosome. \u003cem\u003eRS1\u003c/em\u003e encodes for the protein RS1 important for retinal adhesion (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe median age at onset of symptoms for patients with XLRS is 4 years (but can start as young as 1 year old), and the median age at diagnosis is 7 years (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). By 12 years old, patients with this disease usually have mild visual impairment (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), and would typically possess low vision by the time they are about 25 years old, since visual acuity declines annually (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Approximately 13% of patients experience vitreous hemorrhage at a median age of 9.4 years and about 9% experience retinal detachment at a similar age, which contributes to temporary or permanent vision loss in this disease (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGene therapy treatments in animal models with XLRS show promise for treating this disease using adeno-associated viruses (AAVs) as delivery vehicles (\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). However, two phase I/II clinical trials testing gene augmentation therapies in XLRS patients did not show significant benefit. In one trial, the intravitreal injection of an AAV8 gene delivery vector containing the \u003cem\u003eRS1\u003c/em\u003e gene caused ocular inflammation in adult patients with XLRS treated with the high dose (1\u0026times;10\u003csup\u003e11\u003c/sup\u003e \u0026minus;\u0026thinsp;3\u0026times;10\u003csup\u003e11\u003c/sup\u003e vector genomes per eye, vg/eye) (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Antibodies against the AAV8 capsid were observed, the levels of which appeared to be related to the severity of side effects (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). In another open label, phase I/II dose escalation clinical trial using the AAV2tYF-CB-\u003cem\u003eRS1\u003c/em\u003e gene therapy vector to treat XLRS, varying degrees of intraocular inflammation were observed, including blurred vision, cells in anterior chamber, vitreous cells or vitritis, most of which were successfully treated with steroids. Three out of 13 (23%) of the participants in the high dose group (who received 6 \u0026times; 10\u003csup\u003e11\u003c/sup\u003e vg/eye) developed chronic uveitis, an ocular inflammatory condition that required treatment with steroids (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). These observations prompt the question whether certain subjects experience more severe side effects due to their baseline immunity, since uveitis is a condition that involves activation of T and B cells (reviewed in (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e)). In study participants, the duration of topical steroid administration appeared to be associated with the titers of AAV neutralizing antibodies in serum (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Varying levels of neutralizing antibody in response to gene therapy may point to differences in B cell reactivity among the participants. These inflammatory conditions may have masked efficacy in these trials.\u003c/p\u003e \u003cp\u003eTo determine the immune baseline of XLRS patients and identify any abnormality that may interfere with gene therapy administration, a follow-up study was performed in adults with this disease using flow cytometry. Flow cytometry can be used to identify and quantify populations of immune cells by labeling cells in blood or tissues with cell type specific markers. Identification of circulating dendritic cells and their subtypes was of particular interest, since classical myeloid dendritic cells are professional antigen presenters that trigger the adaptive immune response, and the unconventional plasmacytoid dendritic cells are adept at secreting cytokines in response to viral pathogens (reviewed in (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e)). In the presence of pathogens, myeloid dendritic cells migrate to the lymph nodes to present the antigens to na\u0026iuml;ve T cells. The populations of CD11c\u0026thinsp;+\u0026thinsp;myeloid dendritic cells in the peripheral blood of adults with XLRS were significantly smaller than those in age-matched control subjects, whereas their populations of CD123\u0026thinsp;+\u0026thinsp;plasmacytoid dendritic cells were elevated (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Additionally, abnormal cytokine profiles were noted at baseline including elevated serum interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF)-α levels (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). These findings imply that the baseline peripheral immunity of patients with XLRS may be altered, but the functional implications of these differences are unclear. The XLRS subjects included in that study ranged from 23 to 72 years of age (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), and the immunity of children with XLRS was not investigated. Therefore, it is unclear if these differences in immune cell populations identified by flow cytometry were caused by worsening ocular pathologies in adult subjects during disease progression.\u003c/p\u003e \u003cp\u003eA clinical study was initiated to investigate the baseline immune status of patients with XLRS from 0\u0026ndash;100 years, for which the recruitment is onging. In this current report, initial flow cytometry findings from the cohort aged 0\u0026ndash;7 including five children with XLRS and five age-matched control subjects are presented.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatient recruitment\u003c/h2\u003e \u003cp\u003eThe research was conducted in accordance with IRB #201911037 approved by the Institutional Review Board at the University of Iowa. Patients with molecularly confirmed XLRS were identified through the pediatric inherited eye disorder clinic. They were offered enrollment in the study and if accepted, asked to give a blood sample. Informed consent was obtained from the parents or guardian. All recruited patients were males due to the X-linked nature of this disease.\u003c/p\u003e \u003cp\u003eAge-matched male control subjects without the diagnosis of a genetic eye disease were recruited from the general pediatric ophthalmology clinic; some control subjects underwent standard of care related surgical procedures and had the option for the blood sample to be obtained while under sedation.\u003c/p\u003e \u003cp\u003eThe blood samples obtained from XLRS patients and control subjects were analyzed by the University of Iowa Hospitals and Clinics Pathology Core Laboratory. The tests performed were complete blood counts with differentials and flow cytometry. For complete blood counts with differentials, whole blood was labeled with specific markers and visualized by XN-20 (Sysmex Corporation). Differentials were performed on a Sysmex SP50 slide stainer and imager using an automated software, Cellavision (Sysmex Corporation). For flow cytometry, peripheral blood mononuclear cells (PBMCs) were isolated via density gradient centrifugation. Antibody labeling and washes were performed following standard procedures established and validated in the Pathology Core Laboratory at the University of Iowa. A 10-color FACSCanto flow cytometer (BD Biosciences) was used and results were analyzed on FlowJo (BD Biosciences) or CytoPaint Classic immunophenotyping software (Leukobyte). Standard flow cytometry panel plus the CD16 and Dendrite Cell Determination analyses were conducted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed using GraphPad PRISM. Comparison between groups were performed using unpaired \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAntibodies\u003c/h3\u003e\n\u003cp\u003eAntibodies used for flow cytometry are as follows: CD4-FITC (BD Biosciences, #340133), CD25-PE (BD Biosciences, #341010), CD3 PERCYPCY5.5 (BD Biosciences, #340948), CD16-PE CY7 (BD Biosciences, #335806), CD19-APC (BD Biosciences, #340722), CD56-APC R700 (BD Biosciences, #657887), CD8-APC H7 (BD Biosciences, #641409), CD127-BV421 (BD Biosciences, # 562436), CD45-V500 (BD Biosciences, # 647450), CD14-BV605 (BD Biosciences, #564054), CD11C-APC (BD Biosciences, #340544).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003ePatient characteristics\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSubjects included in this report are a part of a larger, ongoing study with a target recruitment of 20 subjects with XLRS and 20 controls across different ages. In this report, findings from the youngest cohort including five children with XLRS aged 0-7 years and five age-matched control subjects are presented. The characteristics of these children with XLRS and age-matched controls, including genetic mutations and animo acid changes in those with XLRS, are listed in Table 1. The average ages between the children with and without XLRS are not significantly different (p = 0.7707, unpaired t-test). All recruited subjects are males due to the X-linked nature of this disease.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBlood cell counts\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo determine if there are major differences in the populations of blood cells, complete blood counts with differentials were performed. There are no significant differences in absolute blood counts nor percentages of neutrophils, lymphocytes, monocytes, eosinophils, and basophils between children with XLRS and age-matched controls (Table 2). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFlow cytometric analysis using markers associated with innate immunity\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate populations of lymphocytes in PBMCs, whole blood was collected, PBMCs were isolated and stained with antibodies, and flow cytometry was performed. To enrich lymphocytes in subsequent analysis, flow cytometry data was gated by side scatter and CD45, a marker for cells of the hematopoietic lineage. This gating strategy, hereafter referred to as the lymphocyte gate (Figure 1a), excludes neutrophils, monocytes and granulocytes from subsequent analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo identify lymphocyte subsets related to innate immunity, cell populations were further gated by CD11c, CD16a, and CD56. CD11c is an integrin that plays a critical role in the adhesion of immune cells to endothelial cells prior to extravasation and immune activation. The population of CD11c+ cells includes monocytes, granulocytes, a subset of B cells, dendritic cells, and macrophages. Within the lymphocyte gate, children with XLRS tended to have lower percentages of cells that are CD11c+ (Figure 1b, Table 3). The trend of decrease in the population of CD11c+ cells in children with XLRS mirrors findings made in adult XLRS patients (11). The population of CD11c+ cells were then subdivided and gated by their expression of CD56. The percentage of CD11c+/CD56- cells in children with XLRS was not significantly different compared to that in age-matched controls (p = 0.6128, Figure 1c, Table 3). On the other hand, CD11c+/CD56+ cells are natural killer-like and are important for expansion of ɣ\u0026delta; T cells (12). The fractions of lymphocytes that were CD11c+/CD56+ was 40 % lower in children with XLRS compared to that in controls, but the difference between the groups did not reach statistical significance (p = 0.2200, Figure 1d, Table 3), likely due to the sample size of our study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFound on natural killer cells, a subset of monocytes, macrophages, and dendritic cells, CD16a is key to antibody-mediated cytotoxicity (13) as it binds to IgG with a 10-fold higher affinity compared to CD16b (14). The fraction of lymphocytes that are CD16a+ was significantly lower in blood samples collected from children with XLRS (p = 0.0388, Figure 1e, Table 3). On average, the percentage of CD16a+ cells in blood samples collected from children with XLRS is 66 % lower compared to that in age-matched controls. To further identify populations of natural killer cells, double positive for CD16a and CD56, populations were gated by these markers. About 10 % of lymphocytes in controls are CD16a+/CD56+, compared to only about 3 % in children with XLRS, and the difference between the groups was significant representing a significant (p = 0.0454, Figure 1f, table 3). In children with XLRS, populations of natural killer cells were 67 % lower compared to age-matched controls. Cell populations within the lymphocyte gate from a 3-year-old XLRS child (R4) and an age-matched control without XLRS (C3) are shown (Figure 1g). Remarkably, children with XLRS have fewer natural killer cells in their circulation. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFlow cytometric analysis using markers associated with adaptive immunity\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo quantify lymphocyte subsets belonging to the adaptive immune system, cell populations in the lymphocyte gate were further gated by cell-specific markers (Figure 2a, Figure 2b). B cell counts, which were identified by CD19 expression, showed a trend of elevation in blood samples from children with XLRS, in which the counts were 41 % higher on average (p = 0.2348, Figure 2c, Table 3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIdentified by CD3, T-cells play an important role in defenses against infections. T cells are further subdivided into CD4+ and CD8+ subsets, which are considered helper T cells and cytotoxic T cells, respectively. Of note, neither the fractions nor the absolute numbers of all CD3+ cells within the lymphocyte gate were different between children with and without XLRS (p = 0.9309 for fractions; p = 0.6968 for counts, Figure 2d, Table 3). However, the fraction of CD3+/CD4+ cells, that is the percentage of T cells that are helper T cells, is significantly higher in children with XLRS (p = 0.0333, Figure 2e, Table 3). Representative data is shown in Figure 2f. On the other hand, the fractions of CD3+/CD8+ cells, considered cytotoxic T-cells, are significantly lower in children with XLRS (p=0.0082, Figure 2g, representative data in Figure 2h, Table 3). The concomitant elevation in CD3+/CD4+ T-cells by 26 % and reduction in CD3+/CD8+ T cells by 29 % results in a higher CD4/CD8 ratio in children with XLRS (controls: 1.274 \u0026plusmn; 0.125; XLRS: 2.234 \u0026plusmn; 0.081, p = 0.0002). The elevation of CD4/CD8 ratio in children with XLRS mirrors the findings made in adult XLRS subjects by Mishra et al. (11). The populations of CD3+/CD4-/CD8- cells are not different between controls and children with XLRS (Figure 3a, Table 3). Within the CD4+ subset, T regulatory cells (Tregs) help modulate the immune response and immunosuppression. To determine whether the populations of Tregs are altered in XLRS, CD4+/CD25+/CD127\u0026minus; T cells were analyzed. T cells identified by CD4+/CD25+/CD127low or CD127\u0026minus; are analogous to classical Tregs identified by CD4+/CD25+/Foxp3+ (15). Foxp3 is the master transcriptional regulator for Treg-specific genes (16). Although not statistically significant, both the fractions and the absolute counts of CD4+/CD25+/CD127- cells within the lymphocyte gate show a trend of elevation in children with XLRS (Figure 3b, Figure 3c, Table 3). Populations of CD4+/CD25+/CD127- cells in blood are also non-significantly elevated in children with XLRS (Figure 3d, Table 3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTogether, the data suggests that T cells shift toward a CD4 identify in children with XLRS. As a result of possessing a greater percentage of T cells that are CD4+ and a smaller percentage of T cells that are CD8+, children with XLRS possess a higher CD4/CD8 ratio, mirroring findings in adults with XLRS observed by Mishra et al. (11). The total numbers of T cells or lymphocytes were not different. A trend towards having a greater B cell pool is noted in children XLRS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTogether, these findings suggest that children with XLRS also have an altered peripheral immunity, similar to observations in made adults. \u0026nbsp;\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eApparent reductions in CD16a\u0026thinsp;+\u0026thinsp;and CD11c\u0026thinsp;+\u0026thinsp;cell populations in children with XLRS\u003c/h2\u003e \u003cp\u003eA previous study by Mishra et al. investigated the baseline immune status in adults with XLRS (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e); they found aberrations in levels of CD11c\u0026thinsp;+\u0026thinsp;cells and an elevated CD4/CD8 ratio in adult subjects with XLRS (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). However, it was unclear whether these abnormalities in their peripheral immune cell populations developed over time as their ocular pathology progressed, or whether these alterations were present early. To address this, we have initiated a similar study in XLRS including subjects from different age groups. In this current study, initial findings in the youngest cohort of patients consisting of five children with XLRS and five age-matched controls aged between 0\u0026ndash;7 are reported.\u003c/p\u003e \u003cp\u003eIn terms of markers commonly used to identify innate immune cells, significant reductions in populations CD16a\u0026thinsp;+\u0026thinsp;cells and CD16a+/CD56\u0026thinsp;+\u0026thinsp;cells were identified. Notably, populations of CD16a\u0026thinsp;+\u0026thinsp;cells were reduced by 66% in children with XLRS, and populations of CD16a+/CD56\u0026thinsp;+\u0026thinsp;natural killer cells were reduced by 67% in children with XLRS compared to controls. The reduction in this population may have functional consequences that merits further investigation. The CD16b isoform, on the other hand, identifies neutrophils (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) and granulocytes (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Antibodies against CD16a can successfully distinguish natural killer cells from neutrophils (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Presumably representing cells capable of responding to antibody-coated immune complexes, the functional consequence of this finding is unknown without further study. In our study, children with XLRS demonstrate a trend of having fewer CD11c+, CD11c+/CD56-, and CD111c+/CD56\u0026thinsp;+\u0026thinsp;cells in their lymphocyte fractions. However, these changes were not statistically significant, likely due to the sample size in our study. A reduction in the dendritic cell population could mean impaired antigen presentation and/or cytokine secretion.\u003c/p\u003e \u003cp\u003e \u003cem\u003eA shift towards CD4\u0026thinsp;+\u0026thinsp;T cell identity and elevation of CD4/CD8 ratio in children with XLRS\u003c/em\u003e \u003c/p\u003e \u003cp\u003ePreviously, an elevation of CD4/CD8 ratio was reported in adult subjects with XLRS at baseline when compared to that in age-matched controls (XLRS base-line: 3.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9; control: 2.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). This finding opened the line of investigation whether an altered adaptive immunity could have impacted gene delivery in XLRS (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Similarly, we found that children with XLRS also had a higher number of CD4\u0026thinsp;+\u0026thinsp;T cells and a lower number of CD8\u0026thinsp;+\u0026thinsp;T cells, resulting in an elevated CD4/CD8 ratio compared to age-matched control subjects. The fact that this abnormality is present early in XLRS suggests that it could be a primary feature of the disease, and not a consequence of advanced ocular pathology. CD4/CD8 ratio signals the balance between CD4 helper T cells and CD8 cytotoxic T cells. It is a prognostic marker in many diseases, including human immunodeficiency virus (HIV)-associated immune deficiency. In HIV, there is typically an inversion (decrease) in the CD4/CD8 ratio due to the rapid depletion of CD4\u0026thinsp;+\u0026thinsp;T cells, signaling the loss of ability to fight opportunistic infections (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Conversely, an increase in CD4/CD8 ratio may signal an increased immune competence. In an observational, multi-center, and retrospective study investigating factors contributing to COVID-19 disease severity, patients in the critically-ill group had higher CD4/CD8 ratios compared to those in the non-critically ill group after coronavirus infections (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Furthermore, medications including tocilizumab and corticosteroids (methyl-prednisolone or dexamethasone) were more frequently used in the critically-ill group than in the non-critically-ill group (46% vs 4%, p\u0026thinsp;=\u0026thinsp;0.0062 and 85% vs 36%, p\u0026thinsp;=\u0026thinsp;0.0122, respectively). Taken together, these studies support the notion that possessing an elevated CD4/CD8 ratio, as is the case in children with XLRS, could predispose the subject to experiencing more severe inflammation when faced with viral infections or when undergoing treatments utilizing viral vectors. These findings could have implications for gene therapy development for this disease, such as the need for additional immunosuppressive treatments. However, these conjectures need to be further tested.\u003c/p\u003e \u003cp\u003eIn the context of viral vector-based gene delivery, the functional consequences of these immune alterations in XLRS are not known without further study. Changes in populations of peripheral immune cells may imply dysfunction, but not necessarily so. However, our report provides additional evidence that both the innate and adaptive immune systems are abnormal in children with XLRS. In the clinical gene therapy trial, elevation in AAV8 capsid-specific antibodies were observed in adults with XLRS after AAV8-mediated gene therapy, and the antibody levels were not the same among individuals receiving the same dose (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). In another clinical trial for treating XLRS, 23% of the study participants in the high dose group developed chronic uveitis (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), a condition related to activation of T and B cells (reviewed in (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e)), but others in the high dose group did not develop this condition. In our study, children with XLRS showed a trend of having greater populations of B cells in their blood, but the statistical power was limited by the sample size of our study. Determining how immune baseline measurements relate to outcomes would be of particular interest. Interestingly, \u003cem\u003eRS1\u003c/em\u003e expression is a prognostic marker for lung adenocarcinoma, and \u003cem\u003eRS1\u003c/em\u003e gene expression was lower in tumors (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). A previously unknown role of the \u003cem\u003eRS1\u003c/em\u003e gene in immunity may be emerging.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eAlterations in immune cell populations belonging to both the innate and adaptive axes were found in peripheral blood collected from children with XLRS compared to age-matched controls. Some of these findings mirror observations made in adults with XLRS. Together, this supports the notion that aberrations in the peripheral immune compartment are a direct consequence of loss of \u003cem\u003eRS1\u003c/em\u003e function and not secondary to the worsening ocular pathologies in this disease. However, to demonstrate that this is a perturbation present since birth, this conclusion would be further supported by samples from newborn children with XLRS, or longitudinal studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe research was conducted in accordance with Declaration of Helsinki and with IRB #201911037 approved by the Institutional Review Board at the University of Iowa. Informed consent was obtained from the parents or guardian. Clinical trial number: not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis manuscript does not contain individually identifiable information.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAvailability of data and materials\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAuthors do not have competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe study was supported by Chakraborty family fund (PI: Drack), and Knights Templar Eye Foundation career starter grant (PI: Hsu). The funders do not have a role in experimental design or data analysis. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAuthors' contributions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAVD conceptualized the study. GDV, STS, CS, AD and AVD recruited and consented patients. SIS, YH and GDV interpreted the flow cytometry results. YH also analyzed the data, generated the figures and wrote the manuscript. BL, GDV and CRF assisted in data interpretation and scientific visualization. \u0026nbsp;YH, BL, GDV, STS, CS, SS, CRF, AD and AVD critically revised the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAcknowledgements\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to acknowledge funding sources including the Chakraborty Family Fund (PI: Drack), Knights Templar Eye Foundation Career Starter grant (PI: Hsu), and Ronald Keech Professorship.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWu WW, Wong JP, Kast J, Molday RS. RS1, a discoidin domain-containing retinal cell adhesion protein associated with X-linked retinoschisis, exists as a novel disulfide-linked octamer. J Biol Chem. 2005;280(11):10721-30.\u003c/li\u003e\n\u003cli\u003eHahn LC, van Schooneveld MJ, Wesseling NL, Florijn RJ, Ten Brink JB, Lissenberg-Witte BI, et al. X-Linked Retinoschisis: Novel Clinical Observations and Genetic Spectrum in 340 Patients. Ophthalmology. 2022;129(2):191-202.\u003c/li\u003e\n\u003cli\u003eOu JX, Vijayasarathy C, Ziccardi L, Chen S, Zeng Y, Marangoni D, et al. Synaptic pathology and therapeutic repair in adult retinoschisis mouse by AAV-transfer. J Clin Invest. 2015;125(7):2891-903.\u003c/li\u003e\n\u003cli\u003eZeng Y, Takada Y, Kjellstrom S, Hiriyanna K, Tanikawa A, Wawrousek E, et al. \u003cem\u003eRS-1\u003c/em\u003e gene delivery to an adult \u003cem\u003eRs1h\u003c/em\u003e knockout mouse model restores ERG b-wave with reversal of the electronegative waveform of X-linked retinoschisis. Invest Ophthalmol Vis Sci. 2004;45(9):3279-85.\u003c/li\u003e\n\u003cli\u003eVijayasarathy C, Zeng Y, Marangoni D, Dong LJ, Pan ZH, Simpson EM, et al. Targeted Expression of Retinoschisin by Retinal Bipolar Cells in XLRS Promotes Resolution of Retinoschisis Cysts Sans RS1 From Photoreceptors. Invest Ophthalmol Vis Sci. 2022;63(11).\u003c/li\u003e\n\u003cli\u003eHassan S, Hsu Y, Thompson JM, Kalmanek E, VandeLune JA, Stanley S, et al. The dose-response relationship of subretinal gene therapy with rAAV2tYF-CB-hRS1 in a mouse model of X-linked retinoschisis. Front Med (Lausanne). 2024;11:1304819.\u003c/li\u003e\n\u003cli\u003eCukras C, Wiley HE, Jeffrey BG, Sen HN, Turriff A, Zeng Y, et al. Retinal AAV8-RS1 Gene Therapy for X-Linked Retinoschisis: Initial Findings from a Phase I/IIa Trial by Intravitreal Delivery. Mol Ther. 2018;26(9):2282-94.\u003c/li\u003e\n\u003cli\u003ePennesi ME, Yang P, Birch DG, Weng CY, Moore AT, Iannaccone A, et al. Intravitreal Delivery of rAAV2tYF-CB-hRS1 Vector for Gene Augmentation Therapy in Patients with X-Linked Retinoschisis: 1-Year Clinical Results. Ophthalmol Retina. 2022;6(12):1130-44.\u003c/li\u003e\n\u003cli\u003eZhu L, Chen B, Su W. A Review of the Various Roles and Participation Levels of B-Cells in Non-Infectious Uveitis. Front Immunol. 2021;12:676046.\u003c/li\u003e\n\u003cli\u003eFitzgerald-Bocarsly P, Dai JH, Singh S. Plasmacytoid dendritic cells and type I IFN: 50 years of convergent history. Cytokine Growth Factor Rev. 2008;19(1):3-19.\u003c/li\u003e\n\u003cli\u003eMishra A, Vijayasarathy C, Cukras CA, Wiley HE, Sen HN, Zeng Y, et al. Immune function in X-linked retinoschisis subjects in an AAV8-RS1 phase I/IIa gene therapy trial. Mol Ther. 2021;29(6):2030-40.\u003c/li\u003e\n\u003cli\u003eLi W, Okuda A, Yamamoto H, Yamanishi K, Terada N, Yamanishi H, et al. Regulation of development of CD56 bright CD11c + NK-like cells with helper function by IL-18. PLoS One. 2013;8(12):e82586.\u003c/li\u003e\n\u003cli\u003eYeap WH, Wong KL, Shimasaki N, Teo EC, Quek JK, Yong HX, et al. CD16 is indispensable for antibody-dependent cellular cytotoxicity by human monocytes. Sci Rep. 2016;6:34310.\u003c/li\u003e\n\u003cli\u003eBruhns P, Iannascoli B, England P, Mancardi DA, Fernandez N, Jorieux S, et al. Specificity and affinity of human Fc\u0026gamma; receptors and their polymorphic variants for human IgG subclasses. Blood. 2009;113(16):3716-25.\u003c/li\u003e\n\u003cli\u003eYu N, Li X, Song W, Li D, Yu D, Zeng X, et al. CD4(+)CD25 (+)CD127 (low/-) T cells: a more specific Treg population in human peripheral blood. Inflammation. 2012;35(6):1773-80.\u003c/li\u003e\n\u003cli\u003eFontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4\u0026lt;SUP\u0026gt;+\u0026lt;/SUP\u0026gt;CD25\u0026lt;SUP\u0026gt;+\u0026lt;/SUP\u0026gt; regulatory T cells. Nat Immunol. 2003;4(4):330-6.\u003c/li\u003e\n\u003cli\u003eVictor AR, Weigel C, Scoville SD, Chan WK, Chatman K, Nemer MM, et al. Epigenetic and Posttranscriptional Regulation of CD16 Expression during Human NK Cell Development. J Immunol. 2018;200(2):565-72.\u003c/li\u003e\n\u003cli\u003eSelvaraj P, Carpen O, Hibbs ML, Springer TA. Natural Killer Cell and Granulocyte Fc-Gamma Receptor Iii (Cd16) Differ in Membrane Anchor and Signal Transduction. J Immunol. 1989;143(10):3283-8.\u003c/li\u003e\n\u003cli\u003eLiao B, Tumanut C, Li L, Corper A, Challa D, Chang A, et al. Identification of novel anti-CD16a antibody clones for the development of effective natural killer cell engagers. MAbs. 2024;16(1):2381261.\u003c/li\u003e\n\u003cli\u003eObeagu EI. Immunological Equilibrium: Unpacking CD4/CD8 Ratios in HIV. Infection. 2024;16:17.\u003c/li\u003e\n\u003cli\u003ePallotto C, Suardi LR, Esperti S, Tarquini R, Grifoni E, Meini S, et al. Increased CD4/CD8 ratio as a risk factor for critical illness in coronavirus disease 2019 (COVID-19): a retrospective multicentre study. Infect Dis (Lond). 2020;52(9):675-7.\u003c/li\u003e\n\u003cli\u003eSmith JR, Stempel AJ, Bharadwaj A, Appukuttan B. Involvement of B cells in non-infectious uveitis. Clin Transl Immunology. 2016;5(2):e63.\u003c/li\u003e\n\u003cli\u003eZhang T, Cheng G, Chen P, Peng Y, Liu L, Li R, et al. RS1 gene is a novel prognostic biomarker for lung adenocarcinoma. Thorac Cancer. 2022;13(12):1850-61.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1. Subject characteristics of children with XLRS and age-matched controls.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cimg 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th7kYwThD7e6Q8EZsWF02zaGYeD48eM4efKkuS6QJEkYHR3FXXfd5Xu9rc1Ot/VMbD3IxwjCH251h4IzYsMg23QH0jPRacjHCMIfbnWHxpwRBEEQBEFsIig4IwiCIAiC2ERQcEYQBEEQBLGJoOCMIAiCIAhiE0HBGUEQBEEQxCaCgjOiZ8nlcggEAn25ybuVTCaDgYEBlEolUdQxuG4DgQAGBgZEcQ1LS0vm+aFQSBQ3hWEYmJmZaYtdc7lczcLDiUQCgUAAiUTClk5sHIZhYH5+HqFQyPQj7ktOm9qD7Ej0ORScET2JYRjYv38/otFo365PxolEIti5c2dNkNEtKpUKlpaWxGQbTzzxhJjkm4985COYnZ0Vk5umWCxi9+7dXQ1qieYpFou44YYbMDk5ibW1NcTjcWiahng8jtOnT2N8fBwjIyMwDEPMShB9CwVnRE9y/Phx6LqOe++9VxT1JQ888ACy2axrK0KnaRR8ZTIZSJIkJvvixz/+sZjkiwsXLohJAICpqSkwxjA1NSWKiC5TLBaxZ88e6LqOdDqNtbU1HD58GFNTUzh8+DDOnj2LaDSKfD7fcossQfQSFJwRPUepVML09DSi0SiGhoZEcV+ya9cuqKqKWCzW1RYEVVUhSVLdoHBpaQmVSqUvt74iOsutt96KSqWCVCrl6D+Dg4N46KGHIMsy8vl8XT8kiH6CgjOi5zh+/DgA4PbbbzfTisUiAoEAgsGg5czXMAzDUZ7L5RCJRDAwMGDKE4mEawAknh8IBDAyMuI47oWPnykWiwgGg+b1eTfb0tKSbYzNwMAAIpGI61ir/fv3o1Kp4MknnxRFHSUSiaBSqaBYLIoioNqqJkmSzR4cPnbNST+oro4dCARs/89mswCA3bt3IyCMKSyVSojFYqY+uU7FoDWRSGD37t0AgGw2i4BlLFy9sUqZTAYjIyM1thX9wVqupaWlmjyNuoGJX+la13XIsoyJiQlRbDI4OIj77rsPiqKIIpNEImGzQSQScfRXwzBqzuX1Tjzfr435M0I8l/udWL+d7qnec4DYIrA6NBD3BP1Qhn7Fr20kSWKSJInJTJZlBoAVCgVRxNLpNAPANE2rSQPAotEo0zSNqarKADBFUVi5XHa8hiRJLB6PM03TWDweN383Go3azufXkiSJqarKNE1j4XDYdi1Zls1rRaNR835WVlZs12KMsXK5bN5bM/jR88rKCgPAVFVli4uLDACLx+PiaYxV7RGNRm15ODzNqncrvLwcTdNs+tQ0jem6zhhjrFAoMEmSbDJN05iiKKYuOSsrK6Y+ZVlmmqaxdDpt/obTPVnP5zbh9yL6Ay8X/21uX6sNFxcXbdfvZ6w29ArXlZtfNYLbUfQJa73jvsOq9Ue0F6+T3GbW8/3YmNcV6/3w/PyvtW5b74n7aTweN8vEfZboX+BSd5xTq7hl6iX6oQz9ih/b8AcmD3KspFIpBpeHPX8A84evruuuD1h+HetvlMtl84EpBn9WmfUFzl8STvfKX/piAJhKpZgkSTWBA4c/yMV89WhFzzzQkiTJFvxweJC5uLhYk4f5CM6YRW9igMrTU6mULd0qs9rS6X6YS3DG08QgjFmCCGvwza8Nhxco9x/xd/sZ0YZecLJZM3CbwaFO8vruZGOn+shl1mdHszau94xw+/Di6aqq2vyuXC7X/dgk+ge3ukPdmkRP8eyzzwIA3v3ud4sivP/97wcALCws2NJLpRIWFhagqqo5Ro13jUajUYyNjdnOn5iYgCzLWFhYMLsgX3nlFciyjGg0iuHhYdv5g4OD2LlzJwDgzJkzNhkA/NEf/ZGYBF3XxSSg+tvr6+uug9VlWQZcfqeTRCIR6Lpe0/Xzla98BZIk1eiwU7h1gd18880AgG9+85uiyBNHjhwBqr4zODhokz300EOQJAnJZLKme1OW5ZqxUvz+ePcs4QzXz8UXXyyKmsKpTt51110AgKeeespMO3/+PCRJcpwJfOONNwIAvv71r4sizzZ+8sknUalUHO+H+5AVwzCQTCYBAHNzcza/GxwcxCc/+UkAwPz8vJlObB0oOCN6ivPnzwMArrjiClGEoaEhKIpSE0TwMVr79+830/hD+IMf/KCZZiUcDgOWvMPDw1hdXcXc3BxKpRJyuRwSiQRmZmYwMjJS90Xs9PJRVRUAcMMNNzS1phcPSnmQ2i24nr74xS+aaYZhYGFhoebF1SmWl5extrYGwzCQy+UwPz+PRCKBUCiE6elp8XTPFItFVCoVKIriOMGkXvAtjmEkvCMGK37Zvn27mOTI4cOHsb6+jqGhIeRyOWQyGSQSCUQiEXzoQx8STzfxauOvfOUrgMszZXBwEKOjo7Y07ktufsc/NmkSxNaEgjOip+BB1eWXXy6KAAAf+9jHACGI+NznPgdJksyHHSxfvE888QQSiUTNwX+HB4OovsSDwSBkWcbu3bsxPT2N2dlZDA4Omi1aTjitwzY3N2cGkrOzs+bg9806EHhsbAySJNlaJXng6vQy6gSGYSAUCuHSSy/F7t27MTk5ienpaRiGUXeweCP4khtii5kV3jLX7aC4n+EB76uvviqKOsbMzAwCgQB2796N8fFxTE9PI5/Pm/fSCnwJGKePMTi09nNfqlQqNc+fRCJhtu5XKhVbPmJrQMEZ0VeIXZulUgn5fB6RSMTx5ZtMJjE9PV1ziC1hpVLJXI8pGo1icXERhUIBjDEsLy97/rrmDA0NYXV1FYVCAfF43AwuFhYWsHv3bszMzIhZNhyxa7PbXZqhUAjZbBaqqiKdTmNlZQWMMayurmLfvn3i6cQm5/rrrwcAPPPMM6KoBv5h1MpCzDMzM5idnYUsy0ilUlhZWUG5XMba2hoeeOAB8fSuoet6zfOHH8TWhYIzoqfgD/RXXnlFFAHV1o9wOGwGEbx1R1zmgQdD5XIZ1Ykxjgcf+3X8+HFzPMnc3BzGxsZs40rEsUheGR4exuHDh7G6ugpd16FpGgBgdnbW9zU7hbVrs9tdmktLS8jn85BlGcvLy4hEIrYWSWsLZ7NcdNFFQAMb8rFLfGwS0To33XQT4DBG1IlsNgtd17G+vi6KPGEYhjnW7NSpU5iYmMCuXbvMD7Z2tN5dcsklQJ1rPf/887b/X3PNNQCAeDxe89wRD2LrQcEZ0VPwsWYvv/yyKDLhA/C/+MUv4stf/jJkWa7pWhwZGQEsXXON4N2c733ve0WR2TrnlWKxiJGRkZrAZmhoCFNTU2bg6BSA8gf8RgQJ1q7NZrs0rQOzOW7rRDnBB/qL43Y4Xl7wbgwPD0OSJOTzecetngzDwOnTpwEAV111lSgmfDI2NgZZlqHret1B74ZhmBM2+ED/ZuHju2RZdhzf1WgHDC/s2bMHqHOtU6dO2f5/5ZVXAi36LtG/UHBG9BS/+7u/C7i87Dk8iDh16hSy2SwOHjwonmJu+xSLxWpmIBqGgZGREQwMDJjjv3iLHR/0az2XTx7wyvDwMHRdx8LCQs1vF4tF5PN5SJJUM+MLgBkEblSQwLs2H3jgAU9dmpdddhkA4PTp07aWKcMw8IlPfMJyZn14K8OpU6dqWrhisZjr7FevHDp0CKhOBBGv/+CDD5qtpk5d44R/Tpw4AQCYnJx0DND4OMNKpYJwOFzzkeUVXl/EyUKoDrjnsyZb4bbbbjNn9Yq/EYvFasaODQ0NIRqNQtd1x+7aTCZjjkMltiDi2hpWGoh7gn4oQ7/i1zayLDsuQmvFuq6QdWFJK9ZFZfmCkdYFIK3rWum6bqbzxSj5ubIsm+sqWdcw4us4OdHot8V1lZhlbbZuL0JrRVxk04pbHq4b6yKbsiwzRVHMddusWNcc0yyL0IqLdWrVRWK5DuGwnhq/13g83pFFaMWycvjvbhVaKWs6nTb93qr7aDRqpofD4Zr159zsyFzsE4/HGYRFpHkd5TKnNfqasbFYP6y/wcvitggt93cxD61z1t+IPsRxTq3ilqmX6Icy9Ct+bcMfyvUWr2z0YOWsrKywcDhsPjh5HqfgqFAo2FYT50FCuVw2gy3rIpb1gjNm+W1+PUmSWDgcrll8lcMXvnS6t3rUuwc36unP6SXDGuSxBjo8mCqXy446KpfLNr3w8pbLZdsLm19H13XXwDWdTpu/yxfRrfdST6fT5suSX8/pvHplZS4v7n6m1bLqus7i8bhN96gGZW7+Xs+Obvax+iG/Pvdjns6DQLdrcNxsLNZrRVHY4uKi6etivSmXy0yz7CSAqq9y3yb6GycfYoyxAPuV0JFAIIA64p6gH8rQr/i1TalUMheEnZubE8VAdX+73bt3I51O9023wMjICCqVCtbW1kRRXfzqmSC8Qj7WmJGREeTzeZTLZeoeJ0zc6g6NOSN6jqGhIWiahmQy6TqA+5Of/CQkSeqbwCyXyyGfz5urhhMEsbnIZDIYGBhwHDvH66+qqhSYEZ6gljNiw2jFNoZh4IYbbsDo6KjZepbJZHDixAmsra1B13WkUinHrX56kVAoBMMwsLq6Kooa0oqeCcIL5GO/eibt2LEDlUoFqqqaCxc///zzWFhYgCRJePrppx0n+hBbF7e6Q8EZsWG0ahvedbmysoJdu3Yhk8lgfHwckiTh0KFDrvtT9hqZTAaxWMz3g71VPRNEI8jHfkWpVMJnPvMZnDp1ypxBLMsyRkdHce+99zou40FsbdzqDgVnxIZBtukOpGei05CPEYQ/3OpOw+CMIAiCIAiC6AxOYVjD4KyOuCfohzL0K2Sb7kB6JjoN+RhB+MOt7tBsTYIgCIIgiE0EBWcEQRAEQRCbCArOCIIgCIIgNhEUnAnkcjkEAoG6x8DAACKRiLkpdiOWlpYQCAQ8n080JpPJYGRkxLRJMBhEIpGo2bSa2FgMw0AikUAwGDRt1ajuiLYdGRlBJpMRT7PhlGdpaUk8zYZhGJifn0coFMLAwIDNl2KxWN17bIZQKFTzDKl3hEIh8RKEC16e1+LRLrs2YmRkxJMtS6USBgYGkEgkRJFJLpdDJBIxyzAwMIBYLOa4CLeVXC6HWCxmq3/cvxvlJTYWmhAgwNfOkmUZBw8eFMUAgJMnTyKfzwNAw+2BisUi9uzZg0qlYq7HRfyKZm3DicViSCaTkGUZ4XAY27ZtM22iKAqWl5dpFW4LfvXcKoZhIBQKmXbZt28fzp8/j2QyiUql4lh3nGx77Ngx6Lruul2XNc/Bgwdtv6FpmuN6d7lcDh/60IdQqVTMdai2b98OAHjqqaeQzWYBAPF4HIcPHxZyN8fMzAy+/vWv29L4QsmyLCMYDNpk119/fcu/2W02yseKxSKmp6fFZNN+qqqKImia5mu9wGbgPqmqKpaXl0WxibWOuPmqdf3GSCSC7du3e1rYdmZmBrOzswCAcDiMd7/73YDg3051kOgurnXHutGmSANxT9BsGRptdsvhm1BLkiSKTFZWVmwbaosb3m51mrUNq25MzTcT5hsUc6LRKAPAotGoLX2r40fP7YBvTB0Oh23phUKBSZLEJEmybezM654syzbblstlc1NocbN7/huqqtry8N8AULN5NP8dACyVStlkHGt+t3Naod6m3b3IRvmYG9y+3aZcLpvPIe6Xbui6btvs3MkXdF1nqL5nCoWCTcafhbIs29KZ5Vno9JxkwrtJrFNEd3HzU+fUKm6Zeolmy+A1OGOMmc4tBl3lcpnF43Gz0rmdt9Vp1jaMMaaqqusDpVwum/omXsOPnttBPB53fKkwy8sjnU6bael0msmy7BgM8ReRGHjzuuX0AuK/IfqKLMs1v+2ENVi0Eo1GG+ZtBAVnnYU/e7vJysqK6VvcL93eI6lUyjyH/3XyBe4n8XhcFDHGmBncWeuYNWhzqhcc3sAg+jfRXdz8lMactYAsy2ISAOD48eOYnZ2FLMsoFArYuXOneArRImNjY2KS2ZVZqVREEeETp7FcjcZ/cQ4fPoz19XXHLhfehfjyyy+baZFIBGtra477oV5++eVAdXwOJ5fLoVKpIBqNOnZjz83NgTFm85VMJgNd16GqasPunF27dkFVVSiKYvvdUqmE8fFxBINBz7ogakkkEuYYMOu4RD6eqpPjR/lvLy0tmWMCBwYGsLS0ZI5jSyQSWFpasvl/JBJBsVgULwcA2L17N3RdRzwex//8n/9TFJvkcjlMTk4C1W7FQ4cOiaeYnD9/HpIk4QMf+IAoAizPvAsXLphpDzzwAADgk5/8pGO94ExMTECWZSiKYuqaj710G4/GdcHlhmHUjGkbGRmh8b9tgIIznxSLRXPc2WWXXWaTbdu2DZqmYW1tzfHFRPhneXkZv/rYqIU/MCRJEkWED2KxGMbHx1GpVBCPx6FpGiqVCsbHxxGLxcTTm+LYsWMAgBtvvFEUOfKFL3wBAMzNpAHg2WefBYCm6tiJEycAAPv37xdFjiwvLyOTydj2RJybm0M0GoWu6xgfHzcHc9PLyB/33HMPpqenMTo6Ck3TsHPnTiSTSYRCoY7r9I477sDa2ho0TcPo6CiuvPJKU3by5Ens3bsXg4OD0DQN0WgUCwsL2LNnj2OApqoqCoWCp/GC0WgUZ8+ebfiBwD9w3MYqr62tAQAuuugioPoM5Ht6vv/977ed68Ta2hoymYwZxPH7efLJJ4Uzf3XtfD4PVVUxNDRkjpdLJpNQFAWappnPiOnpaXz4wx8WL0E0g9iUZqWBuCdotgyNujXL5TJbXFw0m6/dmput8K446ta006xtGhEOhxkcur62On707Da2r1wum77vx58LhYJpJ7c6ZkXXdbNrR5Ik273w9JWVFbaysmJel9+32J3JLN1dfu5dRNd12/giSZKYpml1u5I41K35mg7g0MXc6vhRfl03+G87df1ZxySKv19vnJeVRu8RK359walbkv+uoii2c71SKBRc8/Pf47biuhDfgfwZoShKzXhPohY3P3VOreKWqZdotgzWilnv4A9iL1Bw5kyztqmHdYKG+LDd6vjRMx/L4uSzfGyY+EKtBx/YzA8vLy0eBKL6AhIf9Lxe8Re5qqpM0zQWjUZdB/Pz67UTtyBNvF8rfl/ImxU/OuU6EAMgZhk/6ue6zIOd643lchtryKlXNzidDs54ECXeB7+Wl991g9c70X9lWbY9X+vpkPCOm59St6YLsiybzbS8SZujaRrW19cdpz0T3SeTyWByctKcVl5vnAXhDd5l79SdwseGNeqSsXLhwgWzLimKgmw2i2Aw6Ng9xAmHw9A0DeFwGLquQ1EUxzFeyWQS6XQay8vLmJqawtzcHJ5++mlIkoTJycm6v2Gl3ppZ9dagGhoawtzcHMrlMjRNAwBMT0+33PW7VXjve98rJmFwcNBcBqOT65Jt27ZNTDIZHR0VkwAA+/btAyzd6t2GL8+E6pg1ax2tVx6vhMNhQOjaLBaL0HUdkUjEfL7yIQmzs7OIRCLIZDKuY9WI5qHgzIVgMIipqSnzmJubQ6FQgCRJmJ6ednxJEN2Hj4uqt94P0Rz8AasoiijyzfDwsFmXVldXzTFbt956q3iqyeHDhzE1NYVMJoN0Om2Od+P3d8kllwDV+xQDxeHhYXzqU58CAHzxi1+0yVB92YhcdNFFUFXVdrhN+nHiueeew7lz58RkogF8ssdGUG/MI5+0spnIZDK47rrrXNcJvPrqq4HqQH2/3HnnnQCAz33uc2Yar0O33367mbZr1y6kUikAwMLCAsbHx811+2gMZutQcNYEw8PD5iKY4+PjHf2iI+rDZwklk0kKzNqMdeZXp5ibm4MkSdB13TFQEolEIjVf9HxRTd6SIcJfVNYFYHlrzJkzZ8w0zvDwMJaXl22H20LUnGKxiJmZGQSDQezdu9dcDDeVSuGxxx4TTycI38zPz2N8fByos3jsVVddBVRbvr0ER5FIBKFQyPYuGxoagqIoyOfz5ofQwsICZFmuaUmfmJhAuVxGOp1GOBw26/T09DR27NhBLWktQMFZk0QiEfMBv3//fk8VgGgv4iyhs2fPUmDWRrgueddmp+BLzHgNBnkwdv78eQDAFVdcYfu/F2655RbAMmvTD6VSCfPz8xgZGcF1112H2dlZrK+vIxqNolAoYHV1FRMTE9S97pFXX31VTNoUNONXnSYWi5lDNwqFgmNghmp3MG/xdppxacUwDJw6dQrZbNac7ck5cOAAUL0G79J0+1AZHBw0uzXX19exuLgIRVFQqVRw/Phx8XTCIxSc+cD61U/O132sWwLRVk2dgT/gnVq1+NismZkZUWSDrx/l9AFjGIa5hQz/2p+ZmTHXmnLi5MmTAIBrrrkGsAR3CwsLtvM4f/VXfwVYAjIAuO222yDLMrLZrO+hCfxFyZcVSKfTWF9fx9zcHH0k+OCZZ54Rk0z/kCSpprWmW5w6dUpMAix+6LQ1VCeYmZlpqofgYx/7GFBd78yp7nGOHz+OSqUCVVVrrnnbbbcB1a5NXk95GofXV/EZMTY2hj/90z8FAHNZD6J5KDjzwdDQkLlw4PT0NDXddpFEIkGBWRfgD/gDBw7UPOA/+9nPAgCuvfZaW7obDz74oJhkplkXkN22bRsqlQo+8YlP1PxmJpNBPp+HLMvmorJDQ0Pm2DVx8H2xWDRfaNb1ngYHB/H4449DkiSMj49jfn7elo9jGAYymQyOHDkiijA0NARN06DrOpaXl11bMQhvzM7O1rzguX/UW6C10+Tz+Rr/4H7oFNB0glwuh9nZWc+BGSy9O7quIxQKOb6fMpmMuScpn8RiZXBwEOFwGPl8HidPnjTXNrNyxRVXoFKpmGM7rXz+858HAHPiAuEDcfqmlQbinqDZMjQzBZpPOW50Li2l4UyztrFOr5dlmamq6noQr9Gsnjl8eQhZlpmmaUzTNNPnxeUPuF2sPq7runm+oig11xDXUGOWuiLLMovH40zTNDPNaSuosmXfTf4b1qU03Jb7KBQK5n1IksSi0ah5f+Fw2MzPyyreZ6v4WT5hM+PHx7gOrHrWNM1mTxHuC430xq/pBv9tp2eyuJwSX6KFr6MnCXvCOtHMe6SeL3BdSJJU84yzHk71gusK1f1tuX9br+m0FiCHr2MGl3rkVPes9dWpfhO1uPmpc2oVt0y9RLNlaKZSLS4ums5bz8m5szo9CLYyfm3j5SBeoxV9pNNp8wGM6gNXXDuMuQRnrPoA1ywBWb1rcMTf5IGa24Pe6TfC4XDNvTiRTqdrgjH+IkylUq6/2Sr1Xsi9iB8fs+pA0zTTBvxjwIluBmeqqtoWHOdBvBefaOY9Us8XeDkaHU7lYC7+zetTowCzXC4zSZJsa5uJONU9Hqi55SHswMVPA+xXQkcCgQDqiHuCfihDv0K26Q6kZ6LT+PGxRCKB6elpaJq2qdaMzOVy2L17N1RVxfLysijeMhiGgR07diASiZirFBDtx63u0JgzgiAIgiBs/Pmf/zkqlQomJiZEEdEFKDgjCIIgCALFYhGhUAgjIyOYnZ1FOBz2NAmBaD8UnBEEQRAEAQA4ffo08vk8otEojh49KoqJLkFjzogNg2zTHUjPRKchHyMIf7jVHWo5IwiCIAiC2ERQcEYQBEEQBLGJ2BLdmgRBEARBEJsRpzhrSwRnvV6GfoVs0x1Iz0SnIR8jCH+41R3q1iQIgiAIgthEUHBGEARBEASxiaDgjCAIgiAIYhNBwZlALpdDIBCoewwMDCASiSCXy4nZTTKZDEZGRsw8wWAQiUQChmGIpxI+IP32Dq3YqlQqYWBgAIlEQhS5kslkEAgEGuYxDAPz8/MIhUIYGBiw3V8sFqtbv1uhVCphZmbGphMvzxTCG7lcDrFYzKbfQCCAUCiE+fl5V7/j5/glkUjYfs/tEH/DMAwkEgkEg0HznJGREWQyGdt5TmQyGUQiEVte7ktu+fk7rlH9IDYWmhAgwDe9lWUZBw8eFMUAgJMnTyKfzwMA0uk0IpGITR6LxZBMJiHLMsLhMLZt22bmURQFy8vLGBwctOXZijRrGw7ptzn86rkdtGIrwzAQCoWQz+c9b45dLBaxZ88eVCqVunlyuRw+9KEPoVKpQJZljI6OYvv27QCAp556CtlsFgAQj8dx+PBhIbd/MpkMYrEYKpUKFEXBvn37AADnzp1DMpkEAESj0Z7baHojfYxjGAYefPBBU4+qquLmm28Gqvo9deoUdF2HLMt4/PHHa7YlCgQCLW12nslkcOLECTHZ5PTp06hUKjb7Wn2c+8P58+exsLAAXdddfaFUKiEcDiOfz0OSJIyOjuLd7343AOD555/HqVOnUKlUoKoqHnvsMVsd4++4evWD6B6udYfVoYG4J2i2DCsrKwwAU1VVFNlIpVIMAJMkyZaeTqcZAKYoCiuXyzZZNBplAFg0GrWlb1WatQ0j/frCj57bQSu20nWdKYrCADAATNM08RRHvOThdRwAS6VSopgxxlihUGCSJNU9p1m4PiRJYoVCQRTbyhyPx0XxpmajfMyKqqqmv+m6LooZs/idLMs1PgkPz32/LC4uOv6uWz0ol8umL6TT6RoZ981oNFpTDn4Ozx8Oh20y7v9u9YPoLm51xzm1ilumXqLZMngNzhhjZgVZWVkx0/gDYnFx0XYuq1YYOAR0W5VmbcNIv77wo+d24NdWqVTKrFv8r5cXSTweZ6i+nOvlkWWZweGlJ8KfBbIs29Kj0WjDvCK6rjNUA0KnwIxTKBTM89wCjM3IRvkYp96HgAj3S9GG8PjcbxZd100/Fm3Pbe10zzygE4Mrt4BOxBrEWX2JgrPNhVvdoTFnLSDLsphkMjY2JiaZTcuVSkUUEU1C+u0O4ngxr2NhrDRjq1wuh8nJSaA6ZODQoUM2uRu5XA6zs7OIx+NmV6ETmUwGuq5DVdWa4Qgiu3btgqqqUBQFpVLJTC+VShgfH0cwGPSsi8985jNAtctS7E6zMjw8jHA4DFVVceHCBVHcV/AxWrlczjbmamBgALFYzHVsmBMPPPAAAODhhx927Sbn3H333VAUBefPnxdFNhqNzeJ1ohHxeByVSgXxeNxm+2KxCEVRoKqq4z1ffPHFAIAf//jHZlqpVEIymYQkSXjooYcsZ9cyODiIaDQKVVXx3e9+VxTbcLNFIBBAJBJBsVgUsxCdRozWrDQQ9wTNlsFry5mfL1z+9ezUWrAVadY2jSD9OuNXz9YuoHg8zjRNM1udGn21N8LNVisrK7auGk3TGn7l8xYC3mpSL49bq0kz6Lpu6oaXQdM0x9YPDtebUytiP+DHx7ideEtnNBplmqbZuifr6ZTj1sLZLBCe+41amLj968GvIUmSp7JY4a2B1pYzrrNW6p9TuRrZQpIkz+85ojncfMg5tYpbpl6i2TI0Cs7K5TJbXFw0H7bNjA0Jh8MtV6x+olnbNIL064wfPbt1E5XLZdP3rd35zeLVVvUCLQ6/Fu8yqpcH1RdqK/fOaSZI4+c4yfoBPz7G7QSHYNlr1x2zXMftme0V8RpOQYwVfu/14MGN2zXcsNYzq278Xs+KU7m4DiWH8ZC8frXym4Q7bj5E3ZouZLNZs1nXelx66aXYu3cv1tfXoWma55lc8/PzWFhY8NQcTTQP6be9/Nmf/Rng0E00ODiIT37yk5BlGa+88oolh3faaSt+rVQqVbe7UGTXrl1iUtMMDQ1hbm7OnFVXqVQwPT2NHTt2IJFImF2h1uUxnLqvtjrRaLSmi5n7BZ956QU+M3OzkMvlkM1mIUkS7rzzTlFcl4985CN1u99vvPFGMaktRCKRmnp01113AdVZzET3oODMBVmWoWmaeUSjUVOmaRrW19c9T0POZDKYnJyEJEl4+umn6QHdZki/7YcvFeMUxEQiEaytrTm+NBrRTlsVi0Xcf//9UFUVExMTorhp6q1x6DbuCJYgrVwuQ9M0AMD09DRisRgA4KKLLhJyEFbe+973ikkYHByEqqqAENz6IRQK1diTH53kC1/4AgDg0KFDTfl5LBbDwsICFEXBY489JorrIpaPH+Laam7w5WSIjYeCMxeCwSCmpqbMY25uDoVCAZIkYXp62vNA4FgshvHxcfNlJH6VEK1B+m0/vMVHURRR1BLttJVhGDhw4AAAOK4D1QinAc4XXXQRVFW1HfUm/Yg899xzOHfunJhsK2czg9y3CpdffrmY5Ivnn39eTAIAXH/99TV27Qb8HXHbbbeJIkcMw8DIyAiSyWTDNQBfeOEFMQmoru1mPdpdh4nuQcFZEwwPD5svgvHx8bpfdIZhmAtwtuNlRNgh/XaOds8S7IStzpw5g3w+by4ia20lmJ6eBqqtV2KrAX8xnzlzxkzjDA8PY3l52Xa4LUTNKRaLmJmZQTAYxN69e80Fd1OplK3Vg78kn3vuOUtuZ+bn5zEyMoL5+XlRRDjAu/h4a6/I4cOHa+zaaZaWlsyFhoeGhkRxDaVSybYYrVtgxrtunT4uANSU8+GHHxZPIXoECs6aJBKJmA/4/fv3O34J81Wf+RfQ2bNnW34ZEa9B+u0sXJduL7tm6JStLrvsMtuwA+vB66eqqtA0Dfv37zfz3XLLLQBQdyX3RpRKJTOAuu666zA7O4v19XVEo1EUCgWsrq5iYmLC9nIdHR0FADzxxBOWKznz5S9/uS267xVeffVVMakpdu3aBUmSoOt63Q/mbvLMM88AQN1lXTh8SY18Po9oNIrV1VXHwAyWj4tMJuP47iH6CHGGgJUG4p6g2TI0mq3JhEUFnWaw8OnI4kw3wk6ztuGQfpvDj565jsWZW8xSR7zMVG7VVvVmXrpRL4/bLDg3nK7FZ8zx54SX63hdhLaVpRc2Ej8+xnXr5EflOosUO8F3bPHqZ9wWYprTbE2ndwFfIFa8Bof7faNZwdZ3iZeZqayJmc7MpQz1Zms61RmnaxDtw82HqOXMB0NDQ+bimNPT07YFKhOJRMOmacI/pN/u8LGPfQwAcODAgZov9M9+9rMAgGuvvdaWLrIZbTU4OIjHH38ckiRhfHzctevQMAxkMhkcOXJEFGFoaAiapkHXdSwvL3uaGMHzAMCePXscW3iKxSI+9KEPAQA+9alPbQp9dYPZ2dmabroHH3wQqA6m98LExASi0Sjy+TxCoZDtmWylWCx6sheqrbOo7olprQOGYeATn/iE5cxa6k2oscL3WXXbQ9OJo0ePQlEUJJPJuov1Li0t4Z577hGTiV5BjNasNBD3BM2WoZmvBP4Fzs/lX3uoLoioqqrrQTRvG9KvP5rVM8e6CK2maUyrswgttwtvKWiXrep90bvhJU+hUDDLIkmSueimpmksHA6brRm8rF5aY7zAt5hCtZXH+ps8vd59b1b8+Bi3k1XPmqbZWltF1AbrfLnpNx6Pm9dF1SfFBYHh8NznduF1IB6PM1mWmaIo5vVE+ALljRbFtba+KYpSUy/44dayyHWB6kK1vKzRaNT0bV4ma2sttZxtLpx8iDFahLaGZhzRWrkWFxfNvF4Owr9tvBzEa7Sij3Q6bXupKYriuBE4l/PgrF22qvfScKOZPOl0uiYYkySJqarKUqlU24IyKyvVXRCsL1AeIDbqBtusNLKjE1Y7aZpm2oAHQk40Cs5YtatQDMZQ9d16OobLc1+zfJRwO1mDIxGv7xBe/kZHves4+RLPE4/HHVf1p+BscwEHH2KMsQD7ldCRQCCAOuKeoB/K0K+QbboD6ZnoNH58LJFIYHp6GpqmeV4zkiD6Dbe6Q2POCIIgCIIgNhGbJjgrFovI5XKuAzkJgiAIgiC2Al0LzkqlEmZmZmpmyhSLRXO9oN27d0OWZUQiEdcZKARBEARBEP1MV8acZTIZjI+Pm//n1zQMAzfccAN0XYckSdi5cyfW1tag6zoURcHq6qrlKv5oVxmI9kO26Q6kZ6LTkI8RhD/c6k7HW85KpZIZmEWjUaysrJiy48ePm4FZPp/H8vIynnvuOYTDYeTzec/7VxIEQRAEQfQN4vRNKw3EnuBrJTlNv+dTnUUZX027HVN3rdOL6aCDDjrooIMOOjbT4UTHuzWDwSDW19exvr5uSy+VSpBlGQCg63rN5rAjIyPI5/Mt/347ykB0BrJNdyA9E52GfIwg/OFWdzreranrOnbu3Ckm4/Tp0wAAWZZrAjNUt1khCIIgCILYanQ8OHPjG9/4BgBgdHRUFBEEQRAEQWxZOh6cybKMtbU1MRkLCwsAgPe+972iCACQzWbNbk+CIAiCIIitQseDs9HRUei6jlwuZ6blcjnoug4AeP/73285+1fwWZob0aqWy+UQCATqHgMDA4hEIrYyWTEMA4lEAsFg0MxT73yiOUi/vUUmk8HIyIhpq2AwiEQi4Wktw1KphIGBASQSCVHkSiaTQSAQaJjHMAzMz88jFAphYGDAdn+xWKxj/sTXfLTqpNEzhfBOLpdDLBaz6TcQCCAUCmF+ft7V7/g5fkkkErbfczvE33B6no2MjHharSCTySASidjycl9yy8/fcY3qB7HBiDMErDQQe4JvmipJEkulUiydTpsb3EajUfF0VigUTLnbBrXN0GwZ+P3yzXedDuuGuul02pa/XC6bckVRmKZpLB6Pm2USz9/KNGsbRvr1hR89tws+W1uWZRaPx231R1GUuhuLW23ttCGzE9bnR708Kysrto22o9GoWb/5htYAWDweF7O2hPX5x/1X0zRTT3B5Lm52NtLHOOVy2aZHVVVt+uWbg8uyzAqFgpjdzOOXdDrNVFV1PZzee27PM36vbr6g67qZT5IkFg6HzbKGw2Hzt1RVraljThufExuHW91xTq3ilqlZtOqO99bD6cFsDXrcnLJZmi0Dd9xGlTSVSpkVwwovazgctqXzl4YkSUzXdZtsq9KsbRjp1xd+9NwO0um0a13nL1G3em59+aCJF4mXPLyOw2EZH441yHM7p1m4PiRJcgwOrGVud1DYaTbKx6zwoFpRFNdngPVjQfRJeHju+2VxcdHxd93qgTVoEz84y+WyLdATy8HP4fnFZyUFZ5sLt7rjnFrFLZMfVlZWWDweZ+FwmKVSKUeH4g7XzgdTs2XwGpwxy/1aW/h4K47Tw5dXRLGybVWatQ0j/frCj57bAX9ZLi4uiiJWLpcZHD5uWPXDh9ct/tfLiyQejzNUX8718vBWiUZ+Ym1FtxKNRhvmFeFrNwJw9F1OoVAwz3MLMDYjG+VjnHofAiLcL0UbwuNzv1l0XTf9WLQ9t7XTPfOATgyu3AI6EWsQZ/UlCs42F251p+Njzji7du3C4cOHkclkMDEx4bhUxtNPP41yuYzDhw+Lok2J04SFw4cPY319HcPDw6II27dvBwC8/PLLoojwCOm3u4jjxbyOhbEyNjYmJpn1v1Kp2NJzuRwmJycBAOl0GocOHbLJ3cjlcpidnUU8Hse+fftEsUkmk4Gu61BVtWafX5Fdu3ZBVVUoioJSqWSm811PgsGgZ1185jOfAaq7pDj5Lmd4eBjhcBiqquLChQuiuK/gY7RyuZxtzNXAwABisZjr2DAnHnjgAQDAww8/7PhusXL33XdDURScP39eFNloNDaL14lGxONxVCoVxONxm+2LxSIURYGqqo73fPHFFwMAfvzjH5tppVIJyWQSkiThoYcespxdy+DgIKLRKFRVxXe/+11RbMPNFoHqeN5isShmITqNGK1ZaSDuOO34cmy2DF5bzvx84fIvdmtL21amWds0gvTrjF89W7uA+HixRmNhvMJbksSWs5WVFVtXDe/GrveVz1sIeKtJvTxurSbNoOu6qRteBk3THFs/OFxvTq2I/YAfH+N24i2d0eq4P2v3ZD2dctxaOJsFwnO/UQsTt389+DUkSfJUFiu8NdDacsZ11kr9cypXI1tINFykY7j5kHNqFbdM3UDTtJoHtx+aLUOj4KxcLrPFxUXzYeulC7ZQKLBwOFz3uluRZm3jBum3Pn707NZNVC6X2xIEc3s1esnUC7Q4/Fq8y6heHlRfqK3cO6eZII2f4yTrB/z4GLcTHIJlr113zHKdVuu+eA2nIMYKv/d68ODG7RpuWOuZVTd+r2fFqVxch05DRnj9auU3CXfcfMg5tYpbpmaxvjxR/cJx+4JcWVkxnbIdv9/sNbjjNjr4g7ge1nEmaMPDo99o1jYipF9v+NEz/4J2CmLS6TSTZbnmheoV62SaRsFKvUCLWa5lHbRfLw/3lXbiFqTxlgbrM6Vf8VM2bienAKxcHZPo5br17N0MaHNw1kqrmdvHJg/OnOqlV5zKVc8WjRosiNZw86GOjzkrlUrYs2ePuegsqls67d27F0tLS2aaYRiIRCLYvXu3uQZaPB435d1GlmVommYe0WjUlGmahvX1dUxNTdnyiFy4cMHMrygKstksgsEg9d+3CdJv58jn80B13JVIJBLB2tpawzFbTmQyGUxOTkKSJDz99NOOY228UiwWcf/990NVVUxMTIjipqm3xqHbuCMAGBoawtzcHMrlMjRNAwBMT08jFosBAC666CIhB2HFaSHywcFBqKoKVO3SCqFQqMaeXseLtcIXvvAFAMChQ4ea8vNYLIaFhQUoioLHHntMFNdFLB8/xLXV3ODjdomNp+PB2Wc+8xlUKhWEw2EUCgWUy2WkUilIkoS7774bqD5kd+zYYQZwqqpC1/UNnRgQDAYxNTVlHnNzcygUCpAkCdPT054GAg8PD5v5V1dXEY1Goes6br31VvFUwgek387AB78riiKKWiIWi2F8fNwMzOoNjG+EYRg4cOAAAGBubk4UN8QpgL/ooougqqrtcJr048Zzzz2Hc+fOicm2cjYzyH2rcPnll4tJvnj++efFJADA9ddfX2PXbsDfEbfddpsocsQwDIyMjCCZTEJRFCwvL7sGdS+88IKYBFTfndaj3XWY6CJiU5qVBmJPyLLsOFCTd0csLi7aps27dXf6pdkyNGrC5WNx/DYtu02p3oo0axsvkH5raVbPfLKLoiiiyBdly+KgTmNa6uHWZeV1+AGEusy7hbx2ybr9PqdQKNgWDeV6E5cL4t3EXp5vqVTKvEavgCZ9jFl06/Yc9dqFx33B6T3jBreVmNaubk2+DIbXOmRd404c52mlXvejE07vM6dy1fNzp2sQ7cPNhzrecqbrOsLhsJhsbtt0xx13oFKpIBqN4uzZs47T7jcTkUjE/PLav39/01/CO3fuBKpdckT7If22Dm/p4V2brWAYBkKhkNkacPbs2ZZazDiXXXaZbdiB9eD1U1VVaJqG/fv3m/luueUWAMCJEyfMtGYplUqYn5/HyMgIrrvuOszOzmJ9fR3RaBSFQgGrq6s1ywXxreieeOIJy5Wc+fKXv9wW3fcKr776qpjUFLt27YIkSTXbBG4kzzzzDADUXdaFw5fUyOfziEajWF1ddW0x476dyWSafvcQPYYYrVlpIPaEWzTOLF8eXr9i/dBsGbx8JVgXFRTLxr/2nL58rINcneRbjWZtw0i/vvCjZ/4V79TKxeuIl5nKXloD6lHvi96NenncZsG54XQt7oOoPie8XMfrIrRct34GkW8kfnyM69bJj3hd9jpjn/fEePUzbgsxzamFyeldwFvGxGtw6k2osWJ9l3htDfM605m5lIFazjYXbj7U8ZazRqgeFoPcbAwNDZmLY05PT9sWqOQ8+OCDYpKZFo1GXb+MCG+QfjvLxz72MQDAgQMHar7QP/vZzwIArr32Wlu6SCKRQD6fbzh+ppsMDg7i8ccfhyRJGB8fx/z8vHgKUG3xy2QyOHLkiCjC0NAQNE2DrutYXl729PzieQBgz549ji08xWIRH/rQhwAAn/rUpzaFvrrB7OxszRhAXpe9LkI8MTGBaDSKfD6PUCjk+ExGVcde7IVq6ywAnD592lYHDMPAJz7xCcuZtdSbUGMlFouZPUdex04ePXoUiqIgmUzWXax3aWkJ99xzj5hM9ApitGalgdgTbtE4ayBrF82WoZmvBP4Fbj1X13UzXbFsbGxN8/JltxVo1jaM9OsLP3pmwiK0op7Fr3ZUWxF4S4G1FVOWZaY6bATNj3rU+6J3w0ueQqFglkWSJHPRTU3YOJqXtV0+xbeYEv3XutRQvfverPjxMW4nq541TbO1torwVks3HbnpNx6Pm9eFy3JOcHjuc7vwOsDHFyqKYl5PhI/ZbDQGztr6pihKTb3gh1vLItcFqgvV8rJGLZu88zJZW2up5Wxz4eRDjHVhnTM3g7MGsnbRbBmacURr5bJW9HJ1lXJrBVF6bIBvN2jWNhzSb3P41TOrToCxvtTc9MzlPDjj9cjLUY96Lw03msmTTqdrgjFJkpiqqjWD+tvFSnUXBKv/8gCxUTfYZqWRHZ2w2kmrLjoOSyDkRKPgjFU/4MRgDFXfradjuDz3rc8abidrcCTi9R3Cy9/oqHcdJ1/ieeLxuOOq/hScbS7g4EOMMRZgvxI6EggEUEfsiUAgAE3THNcEqydrF+0oA9EZyDbdgfRMdBo/PpZIJDA9Pd3xdwBBbGbc6k5XgjNVVXHzzTeLIkxPT7vKOK1W2naUgegMZJvuQHomOo0fH6PgjCDc605XgrNWaMfvt3oNojOQbboD6ZnoNH58jIIzgnCvOx0Pzupte+KFVittO8pAdAayTXcgPROdxo+PUXBGEO51p+PB2UbTD2XoV8g23YH0THQa8jGC8Idb3dkSwRlBEARBEMRmxCnO2rTBWSgUQjabbfn3N7IMRH3INt2B9Ex0GvIxgvCHW93Z8B0CCIIgCIIgiNeg4IwgCIIgCGITQcEZQRAEQRDEJoKCM4FcLodAIFD3GBgYQCQScdy82AnDMDAyMkKTE9qEYRhIJBIIBoOmTZqxB9FdMpmM6f+BQADBYBCJRMJ1w2YrpVIJAwMDTS3Jk8lkEAgEGuYxDAPz8/MIhUIYGBiw3V8sFuuYP5VKJczMzNh00uwzhXAnl8shFovZ9BsIBBAKhTA/P+/qd/wcvyQSCdvvuR3ibzg9z0ZGRpDJZGznOZHJZBCJRGx5uS+55efvuEb1g9hgrHs5iTQQdxS3fcuapdlr8H3ErJs9i4d1z7Z0Oi1eoga+gXSz99Lv+NFHuVy2bYysVTcj5vvyebHHVsOPntuFdfP0eDxuqz9Kg03qrbZ22vPPiUKhYPpCvTwrKyu2vRytG59bN5R22nS6FdLptPm73H+16mbV/DfFTeV7gY30MU65XLbpUVVVm375/pOyLNs2AufwPH5Jp9NMddi4nB/c7lb7uj3P+L26+YKu62Y+SZJsG59b94pVVbWmjjntrUlsHG51xzm1ilumbrDRwVmjSppKpcyKUQ/r5ujN3ku/40cfWnWD3nA4bEvnL2VJkhw3+93K+NFzO0in0wwuQRh/iXp5+TTzIvGSx7opu9Mm7kwI8tzOaRauD0mSHIMDa5nbHRR2mo3yMSv8naEoiuszwPqxIPokPDz3/cLfA+LvutUDa9AmfnCWy2VboCeWg5/D84vPSgrONhdudcc5tYpbpmZYWVnxdXDHapVmr8Ed10sl5RVkZWVFFDFWfdhKkmR7YRCv4UcfvJXM6eXGH3Tiw2yr40fP7YC/LBcXF0URK5fLDC4fN6lUyqxb/K+XF0k8Hmeovpzr5eGtEo38hD8LZFm2pUej0YZ5RXRdN58BTr7LKRQK5nluAcZmZKN8jFPvQ0CE+6VoQ3h87jcLfw842Z7b2umeeUAnBlduAZ2INYiz+hL3a7f6QXQXt7rjnFrFLVMzcOfze7RKs9doJjjjLwG34Iw3ZVsfzMRrtFsfvFWNHjp2WtFzOp22fVwoilLzUnOjUeu3U53g9U+SJJZOpz3blOfjXaduefhL3Ev9ZtUyhMNh28uNl0uWZc+68PpCZYyxcDjMVFWteZFvZkQ7eoHbaWVlhWmaZgbNkiS5tgi5wfO6PYutLC4uMkVRalpERb9oFMQ4+a8T4XDY9E0rhUKBKYri6otO7yL+LpEkyZN+4vE4U1XVphencrnZAtXgsJd8sddw8yHn1CpumZqBG9jv0SrNXsOpQjjR6AuXOzt/eLerPP1Eu/XRzAN6K+FXz9YuIB70NBoL4xXrS8bKysqK7cVcL9Di8BYC3mpSL49bq0kz6Lpu6oaXQdO0ui9LrjenVsR+wI+PcTvx4D9aHffHbeSlFYzVaeFsFnQgOLN+bHgpixX+IWFtOeM6a6X+OZWrkS14IwPRftx8yDm1ilumXqLZMjQKzsrlMltcXDQftuLXELMEbtZK5aUibzXapY9CoWB+nbrZbSvjR89u3UTlcrktQTC3V6OXTL1Ai8Ovxb/u6+Xh9bCVe+c0E6Txc5xk/YAfH+N2gkOw3ExLI79Oq3VfvIZTEGOF33s9eHDjdg03rPXMqhu/17PiVC6uQ6chI7x+tfKbhDtuPrSpl9IoFotiUtfIZrM1U6ADgQAuvfRS7N27F+vr69A0DYcPH7blMwwDt956K2RZxtGjR20yor2USiUEAgFcd911WFhYgKqqWF5eFk8jfPBnf/ZnAICHH34Yg4ODZvrg4CA++clPQpZlvPLKK5Yc3pmfn8fCwgIkScJDDz0kipuCXyuVSmF4eFgUu7Jr1y4xqWmGhoYwNzcHXdcRjUZRqVQwPT2NHTt2IJFIoFQqAdWlCzhWXRK/IhqNIhKJ2NK4XySTSVt6PW6++WYxaUPJ5XLIZrOQJAl33nmnKK7LRz7yEei6DlVVa3QDADfeeKOY1BYikUhNPbrrrrsAAE899ZQtnegsHQ/OQqGQ67oy9UgkErjuuuvE5K4hyzI0TTOPaDRqyjRNw/r6Oqampmx5AODBBx+Erus4ceIEPYg7zIULF0z7KIqCbDaLYDC4oUF9v5DP5wGXICYSiWBtbc3xpdGITCaDyclJSJKEp59+uqU6UiwWcf/990NVVUxMTIjipqm3xmG9NaF4kFYul6FpGgBgenoasVgMAHDRRRcJOQgr733ve8UkDA4OQlVVQAhu/RAKhWrsyY9O8oUvfAEAcOjQoab8PBaLYWFhAYqi4LHHHhPFdRHLxw9xbTU3tm/fLiYRG0THg7NsNosdO3Z4rmClUgmhUAjT09OiqKsEg0FMTU2Zx9zcHAqFAiRJwvT0tOMCf5lMBslkEvF43PGlRrSX4eFh0z6rq6uIRqPQdR233nqreCrRBLzFR1EUUdQSsVgM4+PjZmAmfqE3g2EYOHDgAABgbm5OFDfEKYC/6KKLoKqq7ZBlWTzNleeeew7nzp0Tk23l9POh2u9cfvnlYpIvnn/+eTEJAHD99dfX2LUb8HfEbbfdJooc4YuVJ5NJKIqC5eVl16DuhRdeEJMAoKac7a7DRPfoeHAWj8dRqVSwe/duzMzMiGIbmUzGbAGRJAmpVEo8ZUMZHh42XwTj4+M1AeeJEycAALOzszVfLhz+fzEv0Tpzc3OQJAm6rju+fAlvXLhwQUxqCcMwEIvFkEwm2xKYAcCZM2eQz+dRqVQgy7KtrvEPu+np6ZpWA/5iPnPmjJnGGR4exvLysu04ePCgeJqNYrGImZkZBINB7N27F8lkErIsI5VK2Vo9+Evyueees+R2Zn5+HiMjI5ifnxdFhAO8i4+39oocPny4xq6dZmlpCZVKBYqiYGhoSBTXwBsl8vl83cCMd926Pd/Ecj788MPiKUSP0PHg7PDhw1hcXIQkSZidncXIyIj5Zc4xDAORSATj4+OmQ+fz+bZ0VbSbSCRiPuD3799v+xLev3+/rSvUenD4/y+77DIzjWgfO3fuBDoQYGwleODk9rJrBsMwEAqFzNaAs2fPthyYAcBll11WU8f4weunqqrQNA379+83891yyy2A5UPKD6VSyQygrrvuOszOzmJ9fR3RaBSFQgGrq6uYmJiwvVxHR0cBAE888YTlSs58+ctfbovue4VXX31VTGqKXbt2mR9lm+Wj95lnngEA7Nu3TxTVUCwWzXdeNBrF6uqqY2AGy8dFJpOhVth+R5whYKWBuCmsq19LkmROKV9ZWbGtqdLuGSHNlqHRbE0mLCro9X55+YjX8KMPPlvJadZbubqwqZt8q+JHz7yuijO3mLCmWCP4dcRZn16pN/PSjXp53GbBueF0Le6DqD4nvFzH6yK0rSy9sJH48TGuWyc/4nVZXGrFDb5ji1c/47YQ05xmazq9C6w7vzjRaA1MjvVd4mVmKmtipjNzKUO92ZpOdcbpGkT7cPOhjreccYaGhrC6ump2c+7duxcjIyPYvXs3dF2HLMsoFAqOg+w3G0NDQzh06BBQ7ToRWwKJzvPggw+KSWZaNBp1/fIkvPGxj30MAHDgwIGaL/TPfvazAIBrr73Wli6SSCQadtN0m8HBQTz++OOQJAnj4+OuXYeGYSCTyeDIkSOiCENDQ9A0DbquY3l52dPECJ4HAPbs2ePYwlMsFvGhD30IAPCpT31qU+irG8zOztZ00/G6zJ+zjZiYmEA0GkU+n0coFHJ9JheLRU/2QrV1FgBOnz5tqwOGYeATn/iE5cxa6k2osRKLxVCpVBCNRj2PnTx69CgURUEymUQsFqupn5ylpSXcc889YjLRK4jRmpUGYt/wNZT4oTpsztoumi1DM18J/Avcy7m8rMRr+NGHruum3hXLxtHWtE75Uq/iR89MWIRW1LP41c79m7cUWFsxZVlmqsNG0PyoR70veje85CkUCmZZ+Ir0vIzWjaN5WdvlU3yLKdF/eWtIo/verPjxMW4nq541TbO1torwVks3HbnpNx6Pm9dF1SfFBYHh8CznduF1gG9KriiKeT0Rvs5lo0Vxra1vSnWnAKfDrWWR6wLVNTV5WaOWTd55mayttdRytrlw8iHGNmARWmuFtD4AnRywHTRbhmYc0Vq5xIouws8jXsOvPsrVVeCtDyDFYTsW4lf41TNz2b7JSc9czoMzXo+8HPWo99Jwo5k86XS6JhiTJImpqspSqVTbgjIrK9VdEKz+ywPERt1gm5VGdnTCaidN00wb8EDIiUbBGat+wInBGKq+W0/HcHnuW5813E7W4EjE6zvE+i6sd9S7jpMv8TzxeNxxVX8KzjYXcPAhxhgLsF8JHQkEAqgjbopSqYRYLIZsNgtUu54eeughfOlLX8Lk5CRQndH0uc99ri0DhjntLAPRXsg23YH0THQaPz6WSCQwPT0NTdN6YjgLQXQCt7rTlTFnS0tLtiUyFhcXMTc3h8HBQUxMTKBQKJizVfbs2eM6DoQgCIIgCKLf6XhwFolEsHfvXlQqFaiqirNnz2JsbMx2Dl9fiG+BMjk5iUgk4jrQkSAIgiAIol/peHDG99BLpVJ1Z2wNDg5ibm7OXBNtYWEBN9xwg3gaQRAEQRBEX9PxMWcjIyNNjyMrlUoIh8PI5/Mt/347ykB0BrJNdyA9E52GfIwg/OFWdzoenLXCzMwMDh8+LCY3RaDDm9sSBEEQBEH4xSnO2tTBWTvohzL0K2Sb7kB6JjoN+RhB+MOt7nR8zBlBEARBEAThHQrOCIIgCIIgNhEUnBEEQRAEQWwiKDgTyOVyCAQCdY+BgQFEIhHHzYtRXXRXzGM9EomEmIXwiKhLt8PNNkT3yWQyGBkZMW0TDAaRSCRc1zHkm1N7PR8OvzEyMoKlpSXbOaFQqMZP6h2hUMiW3y+GYWB+fr7m90OhEDKZjHg60QRentfi0alnA/fbgYEBBDz6bS6Xc/T1RhSLRTMPLdren9CEAIFcLofdu3dDlmUcPHhQFAMATp48iXw+DwBIp9OIRCI2Od+WRFEUx3Xd9u/fX5NnK9KsbVB9wbphGIZpF13XMTQ0JJ6yJfGj53YRi8WQTCYhyzLC4TC2bdtm1h9FUWrWPiwWi9izZw8qlQqi0Si2b9+Op556Ctls1vF88TcOHjyI8+fPI5lMolKp2LYGmpmZwde//nVb3rW1Nei6DlmWEQwGbbLrr7++5dnixWIRt956q/kbXAfWe3QrVy+xUT5WLBYxPT0tJpvbBKqqKoqgaVpTSzt5YWlpCXv37gWqv3nzzTfj3Llzpl+eOnWq5nmUyWQwPj4OSZIQjUaxbds2HDt2DLquQ1VVLC8v2863MjMzg9nZWUiShIGBAaytrYmnED2Ca92xb7Vpp4G4J2i2DF43eU2lUgzVjXBF+Ia4hUJBFBEWmrVNI/hGx04bc29l2q1nr6TTaYbqhtPiBuLRaJQBYNFo1JbON3BOp9O29HA4zJw2ZuYbNquqavuNQqFgbqTttPkzp96Gz61ivQexPIwxVi6XTT0oiiKKe4qN8jE3UN0AvBuUy2VXOxcKBdM/rfD3jCRJNv8sl8vmc0y8lhVJkpgsy6b/uG3mTmx+3PzUObWKW6ZeotkyeA3OWLWCOFUMnk7Up506isfjDAALh8OiaMvTTj03A/9IWVxcFEWsXC6bLycOf5E5BSpu9ZLXNTH4Y5YA0On3OV6Cs1QqxTRNc/yNevBAs97HQrlcdg1Ie4mN8jE30MXgjH+EiL7J4X5o/VjnHxtOvsmvF4/HRRFjjLHFxUWG6oeN9d9Eb+LmpzTmrAVkWRaTUCqVzH1Eie5QLBbNJv7Z2VlRTLSA01iuZsdJiXvporpdGwBUKhUz7cKFCzaZlauuukpMQi6XM7s/nfLMzc2BMeb4+81w/vx5TE9PY8eOHQ3HEHEymYzZlTkxMSGKTQYHB3HfffdBURRR1PckEglzDFgikUAwGESgOqY3Fot50rNf+G8vLS2ZYwEHBgawtLRkjmNLJBJYWlqy+X8kEkGxWLRd6+WXXwYA3HzzzbZ0zvbt2wFLVyuq2xrKsuzom5FIBIwx1y71z3/+8wCA22+/HWNjY5BlGclkEqVSSTwVsIy1LBaLpo6DwaB5vmEYrvoPtHHsJdEcFJz5pFgsmuObLrvsMjP9pZdeAgBccsklTQ8OJfzBx5wcOnSoZlwH4Z9YLIbx8XFUKhXE43FomoZKpYLx8XHEYjHx9BqWl5fxqw/DWviLQZIkM40HYKdPn66pJ08++SRQrVecZ599FgDaPn5I5M4774SmaUDV1y699FLEYjHXlyEAfO1rXwMAhMNhUVTDxMQEVldXt+w41HvuuQfT09MYHR2FpmnYuXMnkskkQqFQjR+0mzvuuANra2vQNA2jo6O48sorTdnJkyexd+9eDA4OQtM0RKNRLCwsYM+ePTUBmheef/55oPpRAcBXQG4YhhnY7dq1C7D42Je+9CXhbDt79uxBMBiEpmlQFMV8VoZCIfMZqmkaIpEIMpkMBWUbjdiUZqWBuCdotgxu3SeccrnMFhcXza4IsemZd5Og2j2jaRrTNM0832n8zValWds4YR27QXp1xo+e3caLWbvhxO78ZuDdOmJ3DO+eVhTFHIuzsrJidl9au4Z4XVtZWWErKyvmNXl+py4jES/dmpxyucw0TTPvhd+/05i2el26/YgfH7M+K8UuXbcxiV7h13WD/7YsyzXPDf5Mcfp9Xi9kWTbT+Phj8VwOLwt/p/Dra5rGdF1n0WjU9ClZluv6Iv8t6zm6rtfckxXui05DPrgeRJl1vKTbu5BoD25+6pxaxS1TL9FsGawVs94hSZJjJar3sBcr6VanWds4wR88TvomfoUfPfNByU4BWDqdZrIs17xQvWKdTCO+GJnw0rbWN3GCDbe9tV5pmmZ72dUb78Ua1Fc33II06/3xdCf99SN+fIzr3imo4WMS/VyXNRGciR/XzPIOcAt2xLqh67rpC2IwzsdRwvLctwZEfGC/pmksHo+bHz5isMThvy1+ENT7GKgn478nXo8Jk22IzuHmp86pVdwy9RLNlsFaMbVqqxd/4PNK1syD3Ir1geNUGbYazdpGhH8xur3kiV/hR8/cT9sNb3lwCraY5YUgSRKLx+NM0zSzRUyWZVse/tKBQ8uL9cvf6Xc4foIzKzxQFfXFf5uCM3e47kXbcbh9/ehQtIdIPbvzd4BT0Mhc8nK/RjWwsr4zeEAlBmdOv1G2zNYUPyx4oOcUuPHfd5K56bHeBBxWZ6Yp0V7c/JTGnLkQDAYxNTVlHnNzcygUCpAkCdPT000PikZ18C+fKPDd735XFBNNwschRSIRxwHhhD/4WCo/Y2LqwcewSZKEp59+umasWCaTMdcHPHv2LA4fPoypqSlkMhksLi5C13Xs2bPHHIfEx58pilIzXmt4eBif+tSnAABf/OIXbbJ2USwW8Y1vfENMBgDs3LkTAPDqq6+KIkLg8ssvF5O6xo033igmmfCB/F6IRCJYWVlBOBzGwsICpqensbq6isXFRTz88MO2c7dt22b++6GHHrLJBgcH8bnPfQ4AzL8c7scLCwvmBAV+jI+PmzK3sZB8jBqn3gQcdGEsJ1EfCs6aYHh4GHNzcwCA8fFx15Wm3SoH0V74w+uDH/ygKCJagD+024VhGOZCsW6BGQCcOHECAPCnf/qnNS+MsbExRKNRVCoVMyh/97vfDQDYt2+f7VzO1VdfDQA1C8+2QqlUwvz8PEZGRnDdddeZs4Pj8Th0XTfPu/766wEAzzzzjJnmBp9F52WSBbF52bVrFzKZDKo9UlhdXcXY2JgZoPPZnNwvVVWt8XNYgiI+4YzD64+qqo4HXz2g0cQAojeg4KxJIpGI2fq1f//+mtlEgUAAsizXpHPc0onmsO4G4DQdnfCP28vBD4ZhIBQKIZlMmi1iToGZlYsvvlhMAiwtGXzpgiuuuAKoLnXRSQzDQCaTQSQSgSzLmJycRD6fRzgcxuLiItbW1nD48GHbTOGbbroJqLZkNCKbzULXdayvr4uiLcFmbV1sl1/xAJ37K5/d38y7IJPJoFKpIBKJYHl52fF4/PHHAQDHjh0Tszty0UUXAXXuw8+MVKJ9UHDmg7m5OUiSBF3Xcfz4cZuMdwXxr3srpVIJ+XwekiTVNDETzfHcc88BLtuzEK3D/djpAc3XgZqZmRFFNYRCIdetmtxwe1mfO3cOsHQL8a5DtwDor/7qrwAAt9xyiyhqiuPHj2N8fNxcwiCVSqFcLiOTybh+GPD1p3Rdr7v3oWEYOHLkCADgrrvuEsVbAqfWRcMwkM1mN/RZeerUKTEJqC6xAcuzp1gsmvstixiGgWQyCQB4//vfDwAYGhqCLMvI5/OOvSx8T1jrMixf+cpXgAa9BMPDw6bPifvKOjE8PAxJklzv42//9m/FJKKbiIPQrDQQ9wTNloEPBm00CNI6qNM6uN861VpcgoAP9HQahLoVadY2VpwG5RLO+NGz21IazLIMhttAbg63kdM1nKj3m3xwsjj5w23JBT4hQBK2xxHx4kepVKpmNqYX+LMEDoO7mfBMcBrI3Uv48THrM1TULbdrPbvUg1/XDf7b4iB51sBu3EfF94Pb5BNeDnFWqJuvW32Cz67kE5/gshOGFaedUviEACe4HkT/o6U0uoebbZxTq7hl6iWaLYPX4IxZpiGL5/IKyWecWadIiy+RrUyztrHiNUAg/OuZ+7F15rKbH6P68uAvO+vMZFmWmaqqrocVa92JRqNMs8zWdLK39WWmVNcVjFqW0hDPF/ESnLVCOp0270WWZXMGqvUew+Fww5fuZsePj1mDM+5TmqbZ7CnCA41G9uLXdMNrcAbLEi3cD50CfussZG5jazmc7GutX/XeE3zpGTGAcsK6dAe/x3rBGbMsz2Fd0oN/2PDyE53DzTbOqVXcMvUSzZahmeCM72sGhzVk0um06fS8gjZ6UWw1mrWNFf7AcXq4EnZa0bOTH4utCcwhOBNfcPUOkXQ6bdoXlkBNbJXglKvrjlmXtAiHw558o9PBGau2fMTjcZse+T32yzPByY6NsOpes6wbx4MEJ7oZnKmqaltwnPuhU6DFqvmsHxL8Y6EeTvVL9An++2K6G/x8/tuNgjOx/vBy8vebl3ch4R832wTYr4SOBAIB1BH3BP1Qhn6FbNMdSM9Ep/HjY4lEAtPT09A0DVNTU6J4w8jlcti9ezdUVcXy8rIo3jIsLS1h7969iMfjrvt8Eq3jVndoQgBBEARBbEGCwSBGRkYcZ2w+8sgjAIAPfOADoojoAhScEQRBEMQW5ODBg8jn89ixYwdmZmaQSCQwMzODYDCIbDaLaDS6YbNltzoUnBEEQRDEFmRqagrpdBo7d+7E7OwspqenMTs7i2AwiHQ6bS66TnQfGnNGbBhkm+5AeiY6DfkYQfjDre5QyxlBEARBEMQmYku0nBEEQRAEQWxGnOKsLRGc9XoZ+hWyTXcgPRPt4uc//znOnDljO1588UW8+OKL5GME4cBPf/pTfOtb38K3vvUtvPzyy47/dqo7FJwRGwbZpjuQnolm+cd//Edb8MX/res6rrrqKrzrXe/CVVddZf772muvJR8jtiSVSsUx6OJ/z58/jyuuuALvfOc7Hf8ODQ051h0KzogNg2zTHUjPhBsXLlywBV/8+Pa3v10TgPF/O0E+RvQr3//+92tauqwBGGOsJuCy/v3t3/5t8ZI23OoOBWfEhkG26Q6kZ6JSqdQEYC+++CJ+9KMfOQZgv/M7vyNeoi7kY0QvwhhzDLqs/7744osdgy7+d2BgQLxsU7jVHQrOGlAsFnHdddcBAFKpFCYmJsRTCJ/4tY1hGDh+/DiOHTsGXdcBAOFwGHfddVfLCybGYjEkk0msrKy0fK3Ngl89E71HuVx27I48f/58TQD2rne9C0NDQ+IlfEE+RmxGfvazn7kGXfzvO97xjpqAy/r3zW9+s3jZtuJWdyg4a8DMzAxmZ2chSRIGBgawtrYmnkL4xI9tDMNAKBRCPp+HoijYt28fzp8/j2QyiUqlgnQ6jUgkImbzRCaTwfj4OABQcEZsan7wgx84dkf+9Kc/dQzC3vnOd4qXaCvkY8RG8OMf/7gm2LL+e319He985ztrAi7r39e//vXiZbuKW92h4KwBAwMDGBgYwOjoaN+1qGw0fmzDN0sOh8PIZDJmerFYxJ49ewAA+Xy+6RaBUqkERVFQqVQACs6ITcJ3vvOdmgDszJkz+OUvf2kGX9Zg7PLLLxcv0RXIx4hO8IMf/MAx6OJ/f/GLX9QEW9a/b3/728VLbjrc6g4FZ3VYWlrC3r17EY1G8cEPftD8N21p0R782GZmZgbJZBJPP/00hoeHbTLeJemn9WxkZAS6rmNgYAC6rlNwRnSVl19+uWY82JkzZ/Brv/ZrNQHYVVdd1XCQcbchHyP8IAZbYgD25je/uSbgsv4dHBwUL9lzuNYdVocG4p6glTKEw2EGgK2srDDGGJNlmQFguq7bzltcXGQAWDgctqVzCoUCA8AURbGlr6yssHA4zCRJYgCYLMtM0zRWLpdt57FqOVRVZel02jxfVVVTrus6i0aj5j3y60WjUcfrlctlpmmaeb4kSea54rU5zdyvF1qxjROapjEATNM0UVQXnm9xcZGpqmqzeT/Qbj0T/imVSmxxcZF9+tOfZn/yJ3/C3vOe97BLLrmEvf3tb2e///u/z+6++26WTCbZV7/6VfbDH/5QzL5pIR8jRH72s5+xv/u7v2NPPvkk+9znPsc+/vGPsz/+4z9me/bsYUNDQ+z1r389e8c73sF27drFPvzhD7P777+fJZNJtrS0xL75zW+yCxcuiJfsS9zqDrWcuWAYBi699FLIsmyOM+PjzzRNw9TUlO38gYEBVCoVlMvlmmie57NOKLCOb4pGo9i+fTueeuopZLNZKIqC5eVl23UCgQAkSUKlUkE0GoUkSdi2bRumpqbMLj0u2759OwDg5MmTyOfztjJwRkZGTNnBgwdx7tw5ZDIZyLKMfD4PVVWxvLxsnt/s/XrBr23cCAaDTbd65XI57N6922wRDYVCyGazTV1js9NuPRONWVtbcxwTNjAw4Ngd2eqMr42GfGzr8eqrr9Zt9frRj37k2Npl/ftrv/Zr4mW3HK51R4zWrDQQ9wR+y5BKpWpaYXRdZ7zFSCQejzMALJ1Oi6KaFjd+Hd5aY4X/rtgKx89PpVK2dMaY2dpTT2b9Hd5SJP5GoVBwbZVr9n69AJ+2ESkUCmYrp1OLnxvlcpnJssxkWTZb/6jljGiGM2fOsP/+3/87O3z4MLvjjjvYu9/9bvamN72JDQ0Nsb1797J7772X/cVf/AX7m7/5G/bjH/9YzN43kI/1Hz/4wQ/Y6uoq+9KXvsT+/M//nP3H//gf2S233MLe/e53s8HBQXbRRRexa665hu3du5dFo1F25MgR9oUvfIE988wz7Nvf/rZ4OcIFt7rjnFrFLVMv4bcMiqIwOHRhOgU7rE7XJU+3Bi88kItGo7ZzOWIwxyzBmVMXoqqqjgEjc+nqc7o+h59vDXL83K8X/NqGYw0axXv2Ag/oCoWCmUbBGSHyi1/8gn3zm99kJ0+eZH/6p3/KIpEIGx4eZm94wxvYjh072Ic+9CF26NAh9t/+239jp0+fZv/wD/8gXqLvIR/rPV5++WW2srLCHnvsMTY7O8smJyfZBz7wAXb11Vez3/zN32Rvectb2PXXX8/+8A//kP2n//Sf2MMPP8wef/xxls/ne6rLfbPjVnecU6u4Zeol/JTBKaDipNNpV5lTkOLUouYW4HF4HmtLGA9A6lEul9nKygpLpVJM0zTzd2AJztyCSA6XWwMdP/frhUblaUShUGCapjFN08xgWpZlW7DlBrejOD6NgrOtyz/90z+xYrHI0uk0+/jHP8727dvHrrnmGvb617+eXXnllewP//AP2czMDHv00UfZ17/+dfbTn/5UvMSWhXxsc/Hzn/+cnT17lv3v//2/2V/8xV+wT3ziE+zf/bt/x26++WYmyzL7tV/7NXb55Zezm266iY2Pj7NDhw6xubk5tri4yL7xjW+wV199Vbwk0SHc6g6NOXOAjxFrhK7rtiUb5ufnMTk5aRtbxsdBWceiBQIBwDJ2S4SP5bKObQsEAjXjwDiGYeDDH/4wstmsLV1RFKC6tAS/Fh9j5XYtOPyWn/v1gh/b1IPP1nQaY2eFj9GTZRmrq6s2GY05639+9rOf1YwHe/HFF3H27FnbeDDrmDAaG1Mf8rHucuHChZoxXta/P/jBD8yxXeI4L/73DW94g3hZYgNwqzsUnDnAB+fu3LlTFAHVwb66rtcEI6VSCbIsQ1EUrK6umrsLiMtv8GCnEV6DMz64X1VV7N+/H5dffrkZWPB1wdoRnDVC1Ecj/NimEXxiRqFQqFlqg8N14oVmy7QZ6YSee4Gf/OQntiCM//tb3/qWYwB21VVX4XWve514GcIDW9XHOsWPfvSjmoDL+venP/1pTbBl/fuOd7xDvCSxSXGrOxScCfBZiWJAZYUHXU4tNLzlRdd1HD9+HLOzs1hcXMTY2Jh5Dg+mnGZ2uiEGTBy+FpvTvcBhhim/dx5AinC59bf83K8XmrWNF7y0fOVyOTz77LNiMgCYW0LxVsIbb7zR9Tq9Qif0vJk4f/58TQB25swZfO9736sJvq666ipceeWV4iWIFul3H2s3r7zyimPQxWc9vuENb6jb6vVbv/Vb4iWJHsW17oj9nFYaiHuCZsvAB4m7ja/i8PFl4nl8LFM6nWayLDNJkmxyxhiLRqPmOV6By4B3PoC/0WB969gqPiPTaQA/n31p/S0/9+uFZm3DLGPCnCZG8DXa3OReoDFnmxfDMFgul2PHjh1j99xzDwuFQuyd73wnu+iii9jOnTvZH//xH7MjR46w//E//gc7e/asmJ3oIP3iY+3gn/7pn9ja2hr767/+a/aXf/mX7D//5//M/uRP/oS9733vYzt27GBveMMb2Nvf/nb2nve8h0UiETY1NcWOHj3KnnjiCfZ//s//YefPnxcvSfQxbnXHObWKW6ZeopkyWGf/NXq580Hw4sQAHiDwoCgej9vkzPI7kiTVDF4vl8tMURQmSZItQIBLcMYXwLUuB8HhQRWE4MzvUhrN3K8XmrENhwdPTsEoL6+TzCsUnG08P/zhD9lXv/pVlkwm2d13381+//d/n7397W9nl1xyCXvPe97D/uRP/oR9+tOfZouLi6xUKonZiQ2g13ysFS5cuMC++c1vsqWlJZZMJtn999/PPvzhD7Ndu3axd7zjHez1r389GxoaYnv27GF//Md/zD7+8Y+zz33uc+zJJ59kf/d3f8d+9rOfiZcktjBudYe6NS3wAf3ivo1O8O4/OEwM4APTAbiOfeLdp5IkIRKJYPv27bYNvMVuVbduTTgsKItq99z6+joikQiSyWTN2CkxD/9tAKhUKjW/1ez9eqEZ23BKpRJGR0eh67q58Tks3ZFOC+Lyrk5RB0546RbtNfzouRt873vfc+yO/Od//mfH7sgrrrhCvASxSdisPuYHwzAcuxv535/85Cc1XY1ityNBeMW17ojRmpUG4p6gmTLw1i6v3XdOXYZMaM2qx4qwHRJvsXL6fS5zolwus2g0al6Hb8Wk67rZ6iUuneG2fRO/d6ffauZ+vdCMbayI987L57aUB28NE+3kBLWctZ9vf/vb7NSpU+y//Jf/wiYmJth73/te9pa3vIW99a1vZf/f//f/sWg0yh555BH25JNPsldeeUXMTvQAG+1jzfDKK6+wZ599lqXTaaZpGovFYuyDH/wgu/baa9m2bdvYwMAA+1f/6l+xf/Nv/g27++672Wc+8xl28uRJdvr0afb9739fvBxBtIRb3aGWM8IGn2AQj8dx+PBhUdxWyDbdoVt6/ta3vuU4O/JNb3qTrQWMt4q99a1vFS9B9Cjd8rFG/OIXv3Bs7bIOth8YGKjb6nXJJZeIlyWIjuFWdyg424IEg0FIklTT/Ycud+uRbbpDu/VcKpUcg7Bt27Y5Lk9x6aWXipcg+ox2+5gbP/nJT2oCLuvfb3/72zXBlhiAvfGNbxQvSxAbhlvdoeBsC8LX+ZIkCdFoFNu2bcP58+exsLBgLiPR7PgxP5BtuoNfPf/f//t/awKwM2fO4C1veUvNeLCrrroKkiSJlyC2CH59TGR9fb0m4LL+ffXVV12DLv7vgMd1GQliM+BWdyg426JkMhmcOHHCtqsAX8Q2EonYzu0UZJvu0EjPTgHYiy++iHe84x01Adi73vUuXHTRReIliC1OIx/jfO9733MMuvjfQCBQt9XrbW97m3hJguhp3OoOBWfEhkG26Q6BQAC/+MUv8OKLL9YEYGfOnIEsy47dkb/xG78hXoogHAkEAvjlL3+Jc+fO1QRc1r/btm2r2+1Ira/EVsPtPdgwOCMIgiAIgiA6g1MY1jA4qyPuCfqhDP0K2aY7kJ6JTkM+RhD+cKs7tMsvQRAEQRDEJoKCM4IgCIIgiE0EBWcEQRAEQRCbCArO6mAYBubn5xEKhRAIBMwjFAo13HvTytLSEgKBAHK5nCgifLK0tISRkRHTJiMjI1haWhJPI3ySy+VM3RaLRVFsg59Xj0wm41iP5ufnYRiGeDrQhnuw5vdyUP3sLq3aV4R8jOgnaEKAC8ViEbfeeit0XYcsywiHw+ZirXyzb6dNtkWKxSL27NmDSqXSlVX3ewm/trFuwh6NRgHAtImXzc23Gn70nMvlsHv3bgCAoihYXV0VTzHhLyyn3xDr0ejoKLZv325b9FiSJDz66KMYGxuz5W31Hnh+SZKwc+dOy9nOaJqG4eFhMZnwAPkY+RjhD9e6Y9lns4YG4p7ATxkKhYK5ubfTpt58o3G+4bYbKysrtk3C+2kz7Xbgxza6rjNJkpgkSaxcLpvp5XLZ3Ahd13Vbnq2OHz2vrKyYfosGm8bzc0Ss9cgtfyqVMs9ZXFy0yVq9B55fVVVbOtF+RN17oVX7MvIxog8QfYrjnFrFLVMv4acM/CWfSqVEkYk1GBADuHK5zOLxuFmZ+YOBgjM7fmyTSqVcH6LpdJoBYNFoVBRtafzomb90uO9KkuQa9HI/F1EUpWE9YoyxxcVF8zesAXer90Avzu4h6t4LrdqXkY8RfYDoUxwacyaQyWTM5vGJiQlRbDI4OIj77rsPiqKIIhw/fhyzs7OQZRmFQsFTczfhjS9/+csAgBtvvFEU4f3vfz8A4NSpU6KI8MnOnTsRj8dRqVQQi8VEsStLS0vI5/MN6xEAjI2NQVVVVCoVHD9+XBT7vgeiN/BrX/Ixop+h4Ezga1/7GgAgHA6LohomJiawurpasxfltm3boGka1tbWaHxBm+F7gTqN3eNj/3RdF0VEC3z0ox+FJEnIZrOYn58XxY488cQTAICDBw+KIkf2798PADh58qQoAnzeA9E7+LEv+RjRz1BwJlAqlQAAN910kyjyzMTEBA1K3yBUVQWqg4SJ9jA4OIhHH30UAHD//febdaQe/JxrrrlGFDnCW5fz+bwoAnzeA9E7+LEv+RjRz1BwJsBbZi6++GJRRPQQFy5cEJOIFhgbG0M4HPbc7dNsPRoaGhKTamj2HjjZbLZmSQPxSCQSYjaiyzRrX/Ixop+h4ExAkiQxiSAIAEePHt3wbh8/9yBJElRVrXtcccUVYjZiA/Bj33bj5x7Ix4i2I84QsNJA3BM0WwZVVRkcply3Ar8mzda006xtmMuMKStc14VCQRRtWerpyw23WWjWWW98VpuTTfgsOq8+r+t6zXVavQe3/ET7EXXvBTf7eLUv+RjRD4g+xaGWM4Hrr78eAPDMM8+IohqKxSKCwWBTzd9EZ+FdHTQRozN47fYZGRkBADz77LOiyJGXXnoJqC4E2giv90D0Jl7tSz5G9DMUnAnwiQALCwuiqIZsNgtd17G+vi6KiA6h1hnwz7dokWVZFBFtxEu3z+233w4AOHbsmChy5JFHHgEA7Nu3TxQ54uUeiN7Fi33Jx4h+hoIzgbGxMciyDF3X61ZIwzBw5MgRAMBdd90liokOccsttwAA/vZv/1YU4cknnwQAjI6OiiKijYiz2pzYtWsXVFWFrusNB0IvLS0hm81CkiTceeedotgRL/dA9C5e7Es+RvQzFJw5cOLECQDA5OSkY4BmGAZCoRAqlQrC4bDjmltEZ+ALzd5///221jPDMPDAAw8AAO69914znegM1m4fNzRNgyRJmJ6edn15ZjIZ3HHHHQCARx99tO4+tSJe7oHoXbzYl3yM6Fdo43MXMpkMYrEYKpWKbePzc+fOIZPJmIHZ0aNHG1b2UCiEbDZLG58LtGIb2vjcO370zDd0VlUVy8vLohioBsQ7duwwX1xOv1EsFnHgwAFzJXc/m1L7vYdmN6Xev39/zYLShDfIx8jHCH+41h1xhoCVBuKeoJUy6LrO4vG4OSuIH+FwuGY/zXrQbE1nWrHN4uKizS6KorR1hm0/4UfPXmeh8f1MG/1GOp026wE/VFVlqVTKttehlVbvgef3ejjt10p4Q9S9F1q1rwj5GNGLiD7FoZYzYsMg23QH0jPRacjHCMIfbnWHxpwRBEEQBEFsIig4IwiCIAiC2ERQcEYQBEEQBLGJoOCMIAiCIAhiE7ElJgQQBEEQBEFsRpzirC0RnPV6GfoVsk13ID0TnYZ8jCD84VZ3qFuTIAiCIAhiE0HBGUEQBEEQxCaCgjOCIAiCIIhNBAVndTAMA/Pz8wiFQggEAuYRCoWQyWTE021kMhmMjIyYeYLBIBKJBAzDEE8lmsQwDCQSCQSDQVO/kUgEuVxOPJXwSS6XM3Vr3WDeCX5ePTKZjGM9mp+fd60Trd6DNb+Xg/ynu7RqXxHyMaKfoAkBLhSLRdx6663Qdd228fn58+fNTbYVRcHy8nLNxuexWAzJZNKW7+TJk8jn8655tiJ+bGMYBkKhkKnLffv22WySTqdpY2EBP3rmGzoDgKIoWF1dFU8x4S8sp98Q65GfTanh8x6a3ZRa0zQMDw+LyYQHyMfIxwh/uNYdYa9NGw3EPYGfMhQKBSZJEgPguMF5uVxm0WiUobrhthW+Qa6iKDWb7fI80WjUlr5V8WMbTdMYqpvPW+E2kySJ6bpuk211/OhZ3NC53obN/BwRaz1yy59KpcxzxI3rW70Hr5taE60j6t4LrdqXkY8RfYDoUxzn1CpumXoJP2WQZZkBYKlUShSZlMtl8zxrAKeqquNDgFXzAGCSJImiLYkf28TjcSZJEisUCqLIDH6dAuqtjB8985cOf6nVC3qdXlqMMaYoSsN6xBhji4uL5m9YP2havQd6cXYPUfdeaNW+jHyM6ANEn+LQmDOBTCZjNo9PTEyIYpPBwUHcd999UBRFFAFATfM5qnkAoFKpiCLCI4cPH8b6+rpj18D27dsBAC+//LIoInyyc+dOxONxVCoVxGIxUezK0tIS8vl8w3qEal1RVRWVSgXHjx8Xxb7vgegN/NqXfIzoZyg4E/ja174GAAiHw6KohomJCayurtrGOC0vL+NXwXAtpVIJACBJkigi2sCxY8cAADfeeKMoIlrgox/9KCRJQjabxfz8vCh25IknngAAHDx4UBQ5sn//fgDAyZMnRRHg8x6I3sGPfcnHiH6GgjMBHkDddNNNoqhl4vE4ANCA9TZTLBYRiUSg6zpUVcWuXbvEU4gWGBwcxKOPPgoAuP/++806Ug9+zjXXXCOKHOGDqfP5vCgCfN4D0Tv4sS/5GNHPUHAmkM1mAQAXX3yxKGqJ+fl5LCwsQJIkPPTQQ6KY8EGpVEIgEMB1112HhYUFqKqK5eVl8TSiDYyNjSEcDnvu9mm2Hg0NDYlJNTR7D5xsNluzpIF4JBIJMRvRZZq1L/kY0c9QcCbQiS7HTCaDyclJSJKEp59+mpbRaBMXLlyApmnQNA2KoiCbzSIYDDZcr4jwx9GjRze828fPPUiSBFVV6x5XXHGFmI3YAPzYt934uQfyMaLtiDMErDQQ9wTNlqHebEs/8BmEbjMMtzLN2qYRXNeyLIuiLY0fPbvNQrPOeuOz2pxmsfFZdCsrK7Z0N3Rdr7lOq/fglp9oP6LuveBmH6/2JR8j+gHRpzjUciZw/fXXAwCeeeYZUVRDsVhEMBh0bP42DMNcjJa3mDnNMCTax9zcHCRJgq7r1HrWIbx2+4yMjAAAnn32WVHkyEsvvQRUFwJthNd7IHoTr/YlHyP6GQrOBPhEgIWFBVFUQzabha7rWF9ft6XzVeyTySQURcHZs2cpMOsSfNDvhQsXRBHRJrx0+9x+++2AZQZtIx555BEAwL59+0SRI17ugehdvNiXfIzoa8SmNCsNxD2BnzJ4XYSWL1woNqvz5nanXQKI1/BjG97t7KRXvsivm3yr4kfPjbprrN0+XOci3Fb1Vl1nHhYI9XsPjfIT7UPUvRca2aeRfRn5GNEHiD7F6XjLWS6Xa+nYCE6cOAEAmJycdPxi4i1jlUoF4XDYtnRDIpGgPTS7wIMPPigmmWnRaJT03mGs3T5uaJoGSZIwPT3tOlMtk8ngjjvuAAA8+uijTdnNyz0QvYsX+5KPEf1Kxzc+D1Q3i/VLO37fzzUymQxisRgqlYptA/Nz584hk8mYgdnRo0fNym4YBi699FIAgCzLCAaDwlVfg5Z88GebUqmE0dFR6LpubnyOatcGT6Og2I4fPeeqGzqrdZYnMQwDO3bsMF9cTr9RLBZx4MABcyV3P5tS+70Hnl/yuCn1/v37aQ1Cn5CPkY8R/nCrOx0Pzty+ZrwyNTUlJjVFK2UolUo4fvw4Tp06ZVu4MBwO4w/+4A9qKhmvqF7we0/9hF/bGIaB48ePmwEZqoN8Dxw40HAbl62IHz17eWmh+hEzPj4ONPDpTCaDEydOmGtTAYCqqrjllltw2223OQbTrd5DM/UR1VaYVp83WxXyMW+QjxEibnWn48HZRtMPZehXyDbdgfRMdBryMYLwh1vd6fiYMy9s5PgygiAIgiCIzcSGBWeZTAYjIyMIBALYvXs3du/ejUAggJGREWQyGfF0giAIgiCILcGGdGvyxVk5qqoClr3SUJ1xNzc3Z/7fL50qA9E6ZJvuQHomOg35GEH4w63udL3lLJPJmIuzrqysgDGG5eVlLC8vgzGGlZUVKIqCZDKJpaUlMTtBEARBEERf0/WWs1AohNOnT+Ps2bOOs2dgmbq8c+fOujNovBBocSkPgiAIgiCITuEUZ3U9OAsEAojH4zh8+LAosjEzM4PZ2dmWf78TZSDaA9mmO5CeiU5DPkYQ/nCrO13v1gSAbdu2iUk1eDmHIAiCIAii3+h6y1kwGIQkSVhdXRVFNkZGRlCpVLC2tiaKmqITZSDaA9mmO5CeiU5DPkYQ/nCrO11vOTt48CDy+TxisZgoMonFYsjn8zh48KAoIgiCIAiC6Gu6Hpzdeeed5mzMYDCImZkZJBIJJBIJzMzMIBgMmrM577zzTjF7VzEMA/Pz8wiFQggEAuYRCoXqrsVmGAYSiQSCwaCZJxKJ0EK7bYL023lyuZyp22KxKIpt8PPqkclkHOvR/Pw8DMMQTwfacA/W/F4O8p/u0qp9RcjHiH6i692aqL5cP/zhD9vWNbOiqioee+wx19mczeC3DMViEbfeeit0XbdtfH7+/Hkkk0lUKhXHTbYNw0AoFEI+nzc35rbmSafTNXtyblX82Ib02zx+9GzdM1BRlLrDEPgLy+k3xHrkZ1Nq+LwHnt/rptSapmF4eFhMJjxAPkY+RvjDte6wOjQQt0yhUGCpVIppmsY0TWOpVIoVCgXxtJbwU4ZCocAkSWIAWDqdFsWsXC6zaDTKADBFUWwyTdMYABYOh23p/JqSJDFd122yrYof25B+m8ePnldWVhgA89A0TTzFhJ8jYq1HbvlTqZR5zuLiok3W6j3w/Kqq2tKJ9iPq3gut2peRjxF9gOhTHOfUKm6ZWkFV1boVoN34KYMsywwAS6VSosikXC6b51kDuHg8ziRJcgwyeUDnFPBtRfzYhvTbPH70zF86/KVWL+h1emkxxpiiKA3rEWOMLS4umr9RLpfN9FbvgV6c3UPUvRdatS8jHyP6ANGnOF0fc5bNZnH+/HkxedOQyWTM5vGJiQlRbDI4OIj77rsPiqLY0g8fPoz19XXHpuvt27cDAF5++WVRRHiE9Ntddu7ciXg8jkqlUncSj8jS0hLy+XzDegQAY2NjUFUVlUoFx48fF8W+74HoDfzal3yM6Ge6HpxJkoRKpSImbxq+9rWvAQDC4bAoqmFiYgKrq6uexzgdO3YMAHDjjTeKIqINkH47w0c/+lFIkoRsNov5+XlR7MgTTzwBVGdne2H//v0AgJMnT4oiwOc9EL2DH/uSjxH9TNeDs7m5OSSTybozaDaSUqkEALjppptEkW+KxSIikQh0XYeqqti1a5d4CtECpN/OMjg4iEcffRQAcP/995t1pB78nGuuuUYUOcIHU+fzeVEE+LwHonfwY1/yMaKf6XpwduLECUiShMnJSVx66aUIVKc7ux3dhs8gvfjii0VR05RKJQQCAVx33XVYWFiAqqot7xVKvAbpt3uMjY0hHA577vZpth4NDQ2JSTU0ew+cbDZrW87A6UgkEmI2oss0a1/yMaKf6Xpwls1ma7o1s9ms69FtJEkSk3xz4cIFaJoGTdOgKAqy2SyCwWDD9XQIb5B+u8vRo0c3vNvHzz1IkgRVVeseV1xxhZiN2AD82Lfd+LkH8jGi7YgzBKw0EPcEzZZBVVUGhynX7YDPJpRlWRRtSZq1TSNIv8740bPbLDTrrDc+q81pFhufRbeysmJLd0PX9ZrrtHoPbvmJ9iPq3gtu9vFqX/Ixoh8QfYrT9ZazZtiIPv/rr78eAPDMM8+IohqKxSKCwaDn5u+5uTlIkgRd16l1pwOQfjuP126fkZERAMCzzz4rihx56aWXgOpCoI3weg9Eb+LVvuRjRD/T9eDMa997IpGALMticsfhEwEWFhZEUQ3ZbBa6rmN9fV0UucIHpV64cEEUEW2A9Nt5vHT73H777YBlBm0jHnnkEQDAvn37RJEjXu6B6F282Jd8jOhnuh6ceeX5558Xk7rC2NgYZFmGrut1K6RhGDhy5AgA4K677jLT+d5uTjNRDcMwx9FdddVVopjwAOl34xFntTmxa9cuqKoKXdcbfowtLS0hm81CkiTP++l6uQeid/FiX/Ixop/peHAmbkQLANPT0zUzWcRjYWHBU/NzJzhx4gQAYHJy0jFA4/s7VioVhMNhx6UbHnzwQTHJTItGo23ZN3QrQ/rdWKzdPm5omgZJkjA9Pe368sxkMrjjjjsAAI8++mhTdvNyD0Tv4sW+5GNEv9Lxjc9LpRIURWnauVVVbcsmsX7LkMlkEIvFUKlUbBufnzt3DplMxgzMjh49aqvspVIJo6Oj0HXd3Jgb1aZ3niZulr5V8WMb0m/z+NEz39BZrbM8iWEY2LFjh1m3nX6jWCziwIED5krufjal9nsPzW5KvX//fs8LShN2yMfIxwh/uNYdcYaAlQZiX6DB5rLtppUy6LrO4vG4OSuIH+FwuO7+jeVymWmaZu69ieoG6Y32f9tq+LUN6bc5/OjZ6yy0dDpt2qAe6XTanAnND1VVWSqVsu11aKXVe+D5vR7dfC71G6LuvdCqfUXIx4heRPQpTsdbzkRyuRwuu+wyTwsCtoNOlIFoD2Sb7kB6JjoN+RhB+MOt7nQ9OOs2/VCGfoVs0x1Iz0SnIR8jCH+41Z0NCc5KpRK+9KUv4amnnhJFNbiNA/BKp8pAtA7ZpjuQnolOQz5GEP5wqztdD86KxSL27NnjeYJAq7/fiTIQ7YFs0x1Iz0SnIR8jCH+41Z2uB2eRSAQLCwsIh8O29cHccFqmohk6UQaiPZBtugPpmeg05GME4Q+3utP14CwQCNSdttxuOlEGoj2QbboD6ZnoNORjBOEPt7qzIcGZpmmYmpoSRR0hUF34liAIgiAIYrPhFGd1PTgLBoMYHR3F3NycKOoInSgD0R7INt2B9Ex0GvIxgvCHW93p+PZNIgcPHkQymUSxWBRFBEEQBEEQW56ut5zlcjncc8890HUdkUgE27dvF0+x0Wr3ZyfKQLQHsk13ID0TnYZ8jCD84VZ3uh6cNTsGrNXf70QZiPZAtukOpGei05CPEYQ/3OpO17s1NU1r6thIDMPA/Pw8QqEQAoGAeYRCIWQyGfF0VwzDwMjISNOBKeGMYRhIJBIIBoOmTSKRCHK5nHgq4ZNcLmfqttEQBH5ePTKZjGM9mp+fh2EY4ulAG+7Bmt/LQf7TXVq1rwj5GNFPdL3lrNv4LUOxWMStt94KXdchyzLC4TC2bduG8+fPI5lMolKpQFEULC8vY3BwUMxuIxaLIZlMAm1oCewn/NjGMAyEQiHk83koioJ9+/bZbJJOpxGJRMRsWxo/es7lcti9ezcAQFEUrK6uiqeY8BeW02+I9Wh0dBTbt2/H+fPnsbCwAF3XIUkSHn30UYyNjdnytnoPPL8kSdi5c6flbGc0TcPw8LCYTHiAfIx8jPCHa93hO6A70UDcNlZWVtjKyoqY3Bb8lKFQKDBJkhgAlk6nRTErl8ssGo0yAExRFFFsY3FxkQEwD+I1/OhD0zQGgIXDYVs6t5kkSUzXdZtsq+NHzysrKza/1TRNPMXEzbet9cgtfyqVMs9ZXFy0yVq9B55fVVVbOtF+RN17oVX7MvIxog8QfYrjnFrFLVM7SKfTTFEUW8VANdhxCoj84qcMsiwzACyVSokik3K5bJ7ndr+6rjNJkmzlJF7Djz7i8TiTJIkVCgVRZAbMbvbYqvjRM3/p8JdavaDXzbe539erR8zyASNJEiuXy2Z6q/dAL87uIereC63al5GPEX2A6FOcro85Q7Wbb3x8HPl8HgCgqipUVQUA5PN5jI+PIxaLCbm6QyaTMZvHJyYmRLHJ4OAg7rvvPiiKIopMeBkWFhZEEeGTw4cPY3193bFrgM/8ffnll0UR4ZOdO3ciHo+jUqk0VSeXlpaQz+cb1iMAGBsbg6qqqFQqOH78uCj2fQ9Eb+DXvuRjRD/T9eAsk8kgmUxCURSsrKyAMYbl5WUsLy+DMYaVlRUoioJkMomlpSUxe8f52te+BgAIh8OiqIaJiQmsrq46jnFKJBLIZrOYm5vD0NCQKCY6wLFjxwAAN954oygiWuCjH/0oJElCNpvF/Py8KHbkiSeeAKrrGnph//79AICTJ0+KIsDnPRC9gx/7ko8R/UzXg7MTJ05AkiQsLy87bmq+a9cuLC8vQ5IkPPLII6K445RKJQDATTfdJIo8UywWMT09jXA47Bi4Ee2lWCwiEolA13WoquroV4R/BgcH8eijjwIA7r//frOO1IOfc80114giR/hgat6aLuLnHojewY99yceIfqbrwVk2m0U0Gq07w3FwcBDRaBTZbFYUdRz+mxdffLEo8oRhGLj11lshyzKOHj0qiok2UiqVEAgEcN1112FhYQGqqmJ5eVk8jWgDY2NjCIfDnrt9mq1HXlqXm70HTjabrVnSQDwSiYSYjegyzdqXfIzoZ7oenAHAtm3bxKQavJzTCSRJEpOa4sEHH4Su6zhx4kTdAJRonQsXLpjr4SmKgmw2i2Aw2HC9IsIfR48e3fBuHz/3IEmSOa7V7bjiiivEbMQG4Me+7cbPPZCPEW1HnCFgpYHYF7IsN1x+glVn4ciyLCY3TbNlUFWVwWHKtRfS6TQDwOLxuChynOmz1Wm3PvhszXb4TT/hR89us9Css974rDYn3+az6FY8LpGj63rNdVq9B7f8RPsRde8FN/t4tS/5GNEPiD7F6XrL2cGDB5HP5+s2GcdiMeTzec8DPdvJ9ddfDwB45plnRFENxWIRwWDQLMuJEycAALOzszVN2hz+f1opuv3Mzc1BkiTouk6tZx3Ca7fPyMgIAODZZ58VRY689NJLQHUh0EZ4vQeiN/FqX/Ixop/penB25513mrMxg8EgZmZmkEgkkEgkMDMzg2AwaM7mvPPOO8XsHYdPBPCy/EU2m4Wu61hfXweqs4HE7afEbaj4/y+77DLLlYh2wQf9XrhwQRQRbcJLt8/tt98OWGbQNoJP/tm3b58ocsTLPRC9ixf7ko8RfY3YlGalgdg35XLZ7D50OlRVtS0U2Ap+yuB1EVq+cKGXZnWnJvGtjh99cL9x8o9yuWzq2Um+VfGj50bdNdZuHzff5raqt+o687BAqN97aJSfaB+i7r3QyD6N7MvIx4g+QPQpTtdbzlCdjbm8vIxCoYBUKmW2JqVSKRQKBU/7VXYS3j05OTnp+MXE93esVCoIh8O0dMMG8OCDD4pJZlqj2cBE61i7fdzQNA2SJGF6etp1plomk8Edd9wBAHj00UebspuXeyB6Fy/2JR8j+hXa+NyFTCaDWCyGSqVi2/j83LlzyGQyZmB29OhRT5Wdjzvzcy/9ih/blEoljI6OQtd1c+NzVLs2eNpGB/ebDT965hs6q3WWJzEMAzt27DBfXE6/USwWceDAAXMldz+bUvu9h2Y3pd6/fz+tS+gT8jHyMcIfrnVHbEqz0kDcNOl02nXfw2g0ysLhsKcuwmZopQy6rrN4PF6zB2g4HHYthxtOTeJbHb/6KJfLTNM0s/sZ1T1Z63VDb2X86Nlrdw2fodzoN9LpdM1QBlVVWSqVcu2CbvUeeH6vR6OuMcIdUfdeaNW+IuRjRC8i+hSnKy1nvBswn89DURSsrq6KpyAYDELXdaC6dZLXFqlGtKsMRPsh23QH0jPRacjHCMIfbnWnK2POeGAmyzIOHDggigEAjz/+uDl+YGFhAR/5yEfEUwiCIAiCIPqejrecZTIZjI+PIxqN4qGHHmrYGmZtZUun0y33z7ejDERnINt0B9Iz0WnIxwjCH251p+PBWSgUwunTp3H27NmGgRmHD8AcHR1FJpMRxU3RjjIQnYFs0x1Iz0SnIR8jCH+41Z2Od2tmPWx0LjI4OIhIJOJpIViCIAiCIIh+ouMtZ4FAAJqmYWpqShTVJZFIYHp6ui2/TxAEQRAEsRlxinO6EpzF43EcPnxYFNVlZmYGs7Ozbfn9Vq9BdAayTXcgPROdhnyMIPzhVnc6HpyFQiGsra1hbW1NFNUlGAxCkiTHZTeaoR1lIDoD2aY7kJ6JTkM+RhD+cKs7HR9zdsstt0DX9aYG9s/Pz0PXdYyOjooigiAIgiCIvqbjwdltt90GWZYxPj7uuE+lyPz8PCYnJyFJEu68805RTBAEQRAE0dd0PDgbHBzE448/DkmSMDk5iWAwiJmZGSwtLSGXyyGXy2FpaQkzMzMIBoOYnJwEAMzNzWFoaEi8XFcxDAPz8/MIhUIIBALmEQqF6rYELi0t2c4XD7cNegnvlEolxGIxDAwMIBAIYGBgALFYDIZhiKcSPsjlcqa/FotFUWyDn1ePTCbjWI/m5+ddbdbqPVjzezlyuZwtP9FZWrWvCPkY0U90fMwZp1gs4tZbbzW3aHJDlmWcOHECu3btEkW+8FsG6/1aNz4/f/48kskkKpWK6ybbfKapoig1MtDmtyat2GbPnj2oVCqIRqPYvn07nnrqKWSzWUiS1NSaelsBP3rmGzoDcN1yjcNfWE6/IdYjP5tSw+c9NLsptaZpGB4eFpMJD5CPkY8R/nCtO9aNNkUaiH2RTqdZNBo1N6iVJImpqsri8ThbXFwUT28ZP2UoFApMkiQGwHGD83K5zKLRKEN1w20RXrZCoSCKCAt+bFMul03biP7CbRKNRm3pWx0/ehY3dK63YTM/R8Raj9zyp1IpV3u2eg9eN7UmWkfUvRdatS8jHyP6ANGnOM6pVdwy9RJ+yiDLMgPAUqmUKDIpl8vmeWIAxx8ERH386CiVSjEALB6PiyKm6zqDS8C8lfGjZ/7S4b4sSRLTdV08jTGXlxZjjCmK0rAeMcbY4uKi+RvlctlMb/Ue6MXZPUTde6FV+zLyMaIPEH2K0/ExZ71GJpMxm8cnJiZEscng4CDuu+8+KIpiSy+VSqhUKlBV1ZZOtIcvf/nLAOA4WWRoaAiMsbpdE0Rz7Ny5E/F4HJVKBbFYTBS7srS0hHw+37AeAcDY2BhUVUWlUsHx48dFse97IHoDv/YlHyP6GQrOBL72ta8BAMLhsCiqYWJiAqurq7bxYy+99BIA4JJLLkEkEjEHrAeDQSQSCdeBqYQ3+LiyjZ4sspX46Ec/CkmSkM1mPc24BoAnnngCAHDw4EFR5Mj+/fsBACdPnhRFgM97IHoHP/YlHyP6GQrOBEqlEgDgpptuEkWe+OY3vwkA5kDUQ4cOQdM0AMD09DRCoRAFaC2yc+dOGIaBRCKBYDCIAM3W7CiDg4N49NFHAQD333+/WUfqwc+55pprRJEjfDB1Pp8XRYDPeyB6Bz/2JR8j+hkKzgSy2SwA4OKLLxZFTaFpGlZXVzE1NYWpqSmsra0hGo0in8/jwx/+sHg64QE+Dd0wDIRCIRw5cgThcBiapkGWZSSTSdxwww0UoHWAsbExhMNhz90+zdYjLy2hzd4DJ5vN1ixpIB60vM3G06x9yceIfoaCMwFJksSkppiamgJjzHGj94ceegioVmT6KvMP//I9e/YsDh8+jKmpKayuriIajULXdXzkIx8RsxBt4OjRoxve7ePnHiRJgqqqdY8rrrhCzEZsAH7s22783AP5GNF2xBkCVhqIe4Jmy8CXwRCnXLcLfv2VlRVRtOVo1jaFQsGcMeVmHz7ryjoja6vTrJ5ZnVlo1llvfFab0yw2PovOq5/zmbbW67R6D275ifYj6t4Lbvbxal/yMaIfEH2KQy1nAtdffz0A4JlnnhFFNRSLRQSDwZrmb2oV6wzWxRvFxSQ5fFzJmTNnRBHRBrx2+4yMjAAAnn32WVHkCJ9II85+dsLrPRC9iVf7ko8R/QwFZwJ8IsDCwoIoqiGbzULXdayvr5tpgUAAsiy7jntySye8IcsyQHrcULx0+9x+++0AgGPHjokiRx555BEAwL59+0SRI17ugehdvNiXfIzoZyg4ExgbG4Msy9B1vW6FNAwDR44cAQDcddddZjr/KnvyySfNNE6pVEI+n4ckSW3bnmqrMTo6Crjo1zAMZKtLbZB+O4c4q82JXbt2QVVV6LrecCD00tKSaTen9euc8HIPRO/ixb7kY0Q/Q8GZAydOnAAATE5OOgZofLZgpVJBOBy2BQIf+9jHAAAPPPCArXXHMAxz7bRDhw6Z6URz3HvvvQCAWCxWs1Hxgw8+CACIRqO2dKL9WLt93NA0DZIkYXp62vXlmclkcMcddwAAHn300ab2RPVyD0Tv4sW+5GNEv9K1jc83Cr9lyGQyiMViqFQqto3Pz507h0wmYwZmR48eranssVgMyWQSkiSZgQJf9ywajWJubs52/lalFduMj49DkiREIhFs374dJ0+eRD6fd92MfivjR898Q2dVVbG8vCyKgeoHx44dO8wXl9NvFItFHDhwwFzJ3c+m1H7vodlNqffv329bUJrwDvkY+RjhD9e6I84QsNJA3BO0UgZd11k8HjdnBfEjHA7X7Kcpkk6nbfkURWmYZ6vRim1WVlZYOBw29SvLMtM0jWZpOuBHz15noaXTadMG9Uin0+ZMZX6oqspSqZSrzVq9B57f61Fv02uiPqLuvdCqfUXIx4heRPQpDrWcERsG2aY7kJ6JTkM+RhD+cKs7NOaMIAiCIAhiE0HBGUEQBEEQxCaCgjOCIAiCIIhNBAVnBEEQBEEQm4gtMSGAIAiCIAhiM+IUZ22J4KzXy9CvkG26A+mZaCcvvfQSXnjhBbz44ot44YUX8MILL+Cb3/wm+RhBuPCtb30L586dsx087e///u8d6w4FZ8SGQbbpDqRnwg9///d/bwZf1mDsHe94B66++mrzeNe73oXrrruOfIzYsnz729+uCbqsx9vf/nZs377ddrzzne/E9u3bIcuyY92h4IzYMMg23YH0TNTjO9/5Tk1L2Isvvoht27bZgjB+/MZv/IZ4CfIxoq/57ne/WxNwWQOx3/qt36oJuqzHv/gX/0K8pIlb3aHgjNgwyDbdgfRMAEC5XHZsCXv9619vtoBZgzBJksRLuEI+RvQy3//+92sCLushSVJNwGUNxH79139dvKRn3OoOBWcNKBaLuO666wAAqVQKExMT4imET/zaxjAMHD9+HMeOHYOu6wCAcDiMu+66y7YJPfEr/OqZ6E1effVVx5awn/zkJzWtYO9617vwtre9TbxE05CPEZuZH/7wh45BF0/7zd/8zZqAy3q86U1vEi/ZNtzqDgVnDZiZmcHs7CwkScLAwADW1tbEUwif+LGNYRgIhULmJuf79u3D+fPnkUwmUalUkE6naWNhAT96JjY/P//5z2tawV544QX84Ac/qGkFu/rqq3HFFVeIl2gb5GPERmIYhmPQxY83vvGNjkEXT/vN3/xN8ZJdw63uUHDWgIGBAQwMDGB0dBTJZBIrKyvUOtMm/NgmkUhgenoa4XAYmUzGTC8Wi9izZw8AIJ/PY2hoyJJra+NHz8TmwtodyYOxs2fP2lrA+L937NghZu845GNEJ6lUKo5BF0973ete5xh08ePiiy8WL7lpcKs7FJzVYWlpCXv37kU0GsUHP/hB899zc3PiqYQP/NhmZmYGyWQSTz/9NIaHh22yWCyGZDJJrWcCfvRMbAxnz56taQl74YUXEAwGa1rCrr76ajH7hkE+RrTCq6++WhNwWf//y1/+0jHo4mnNjI/cbLjWHVaHBuKeoJUyhMNhBoCtrKwwxhiTZZkBYLqu285bXFxkAFg4HLalcwqFAgPAFEWxpa+srLBwOMwkSWIAmCzLTNM0Vi6XbeexajlUVWXpdNo8X1VVU67rOotGo+Y98utFo1HH65XLZaZpmnm+JEnmueK1Oc3crxdasY0TmqYxAEzTNFG0pWm3nonW+da3vsWWlpbYpz/9abZ//362c+dO9uY3v5kNDQ2xD37wg2x6epp9/vOfZ1//+tfZz372MzH7poN8jKjHhQsX2De+8Q32xBNPsM9+9rPs3nvvZbfddhtTFIW95S1vYRdddBG79tpr2b/+1/+a/Yf/8B/YZz7zGfalL32J5fN53++XXsGt7lDLmQuGYeDSSy+FLMvmODM+/kzTNExNTdnOHxgYQKVSQblcxuDgoE3G81knFGQyGYyPjwMAotEotm/fjqeeegrZbBaKomB5edl2nUAgAEmSUKlUEI1GIUkStm3bhqmpKbNLj8u2b98OADh58iTy+bytDJyRkRFTdvDgQZw7dw6ZTAayLCOfz0NVVSwvL5vnN3u/XvBrGzeCwSB0XaeuZ4F265nwzve///2aVrAXX3wRb3rTm2oG5l999dWbuvulHuRjW5uf/vSnjt2N/N//8A//ULfl67d+67fES24ZXOuOGK1ZaSDuCfyWIZVK1bTC6LputhiJxONxBoCl02lRVNPixq8DgC0uLtrO5b8rtsLx81OplC2dMcZUVW0os/4Ob2ESf6NQKLi2yjV7v16AT9uIFAoFs5XTqcVvq9MuPRPurK+vs5WVFZZKpdjdd9/N3ve+97G3ve1t7C1veQv7vd/7PRaLxdjRo0fZV7/6VfajH/1IzN7zkI/1Nz/72c/YSy+9xJaXl9n8/Dy7//772fj4OHvPe97Dfvu3f5u98Y1vZFdeeSULhUJscnKSHTlyhKXTafY3f/M37Hvf+554OcKCW91xTq3ilqmX8FsGRVEYHLownYIdVqfrkqdbgxceyEWjUdu5HDGYY5bgzKmJV1VVx4CRuXT1OV2fw8+3Bjl+7tcLfm3DsQaN4j0Tr9GqnonX+MlPfsJOnz7N/vIv/5Lde++97AMf+AB75zvfyS6++GL2nve8hx04cID9+Z//Octms+yVV14Rs/ct5GO9zT//8z+zs2fPsieffJL91//6X9nMzAy744472K5du9jll1/O3vCGN7Df+Z3fYaOjo+zf//t/zw4fPswee+wx9swzz7DvfOc74uWIJnCrO86pVdwy9RJ+yuAUUHHS6bSrzClIcWpRcwvwODyPtSWMByD1KJfL5te7pmnm78ASnLkFkRwutwY6fu7XC43K04hCocA0TWOappnBtCzLrFAoiKduaVrV81bkn//5n1mhUGCPPfYYm5mZYX/wB3/AduzYwd74xjeyd7/73ezf/tt/y44cOcL+1//6X6xUKonZtxzkY5ubX/ziF0zXdfbXf/3X7C/+4i/Yxz/+cfZHf/RH7L3vfS+74oor2Otf/3omyzJ73/vex+6880720EMPsc9//vPsa1/7Gnv55ZfFyxFtxK3u0JgzB/gYsUboum5bsmF+fh6Tk5O2sWV8HJR1LFogEAAsY7dE+Fgu69i2QCBQMw6MYxgGPvzhDyObzdrSFUUBqktL8Gvlcjns3r3b9Vpw+C0/9+sFP7apB5+t6TTGbivTbj33G3wjb3HNMHGJiquvvhpXXnmlmJ0gH9sUiDMcxXFf1jFeTuO+iI3Bre5QcObAwMAAAGDnzp2iCACwtrYGXddrgpFSqQRZlqEoClZXV83dBcTlN3iw0wivwRkf3K+qKvbv34/LL7/cHBDP1wVrR3DWCFEfjfBjm0bwiRmFQqFmqY2tSif03IuIG3nzQMy6kbc1GKu3Hx5hh3ys87Syufb27dvxute9TrwksQlwqzsUnAnwWYliQGWFB11OLTShUAjZbBa6ruP48eOYnZ3F4uIixsbGzHN4MOU0s9MNMWDi8LXYnO4FDjNM+b3zAFKEy62/5ed+vdCsbbzA9U8zNl+jE3rezPCNvMX1wiRJqmkJu9plI2+iObaaj3UCp821rUFYK5trE5sX17oj9nNaaSDuCZotA5/15za+isPHl4nn8TFp6XSaybLMJEmyyRljLBqNmud4RRwHxuED+BsN1rdOCOAzMp0G8PPZl9bf8nO/XmjWNswy/s1pYgRfo81NvlXxo+de4Ec/+hH76le/yo4ePcpisRj7vd/7PfaWt7yFve1tb2Pve9/72N13381SqRRbWVlh6+vrYnaijfSrj7WT733ve+xv/uZvWDqdZkeOHGGTk5MsFAqxK6+8kr3xjW9kv/3bv83e8573sPHxcXb//fez+fl5try8zF566aWeWOuO8Idb3XFOreKWqZdopgzW2X+NXu58ELw4MYAHCDwoisfjNjmz/I4kSTWD18vlMlMUhUmSZC5+y+oEZ3wBXFmWa+6ZB1UQgjO/S2k0c79eaMY2HB6cOQWjvLxOsq2MHz1vJs6fP8+effZZ9rnPfY7dc889bHR0lL397W9nAwMDbNeuXWxiYoI98sgj7K//+q9p2v4G0es+1g5+8IMfsNOnT7MvfvGLLJFIsFgsxsbGxtjVV1/N3vzmN7O3vvWt7IYbbmDhcJhNTU2xubk5trS0xF588UX2j//4j+LliC2CW92hbk0LfEC/uG+jE7z7Dw4TA/jAdACuY59496kkSYhEIti+fbttA2+xW9WtWxMOC8oCwLFjx7C+vo5IJIJkMlkzHkzMw38b1X3MxN9q9n690IxtOKVSCaOjo9B13dz4HNXy8jQ/C+L2M370vBFYN/K2dkv+4Ac/qBkPdnWHN/ImmqNXfKwVenlzbWLz4lZ3KDizwGdWet2bkZ8vBj6NxoFxcrkcPvvZz+LUqVOoVCoAYA7qF3+/XnBmGAYefPBBZDIZVCoVM4C69957AcA2ScGa5/jx42ZQw/PwPUSdfquZ+/VCM7axIt47qjNTDxw4YM6SJV7Dr547iRiEvfDCC1hbW7MFXzwY24iNvInm2Iw+1iyVSqUm4LIGYb28uTaxeXGrOxScETZ4YBmPx3H48GFR3FbINt1hI/XMN/IWB+fv2LGjpiXs6k20kTfRHBvpY17xsrm2GHBZg7Be3lyb2Ly41R0KzrYgwWAQkiQ5dv91c7Yj2aY7dEPPL7/8ck1L2Isvvoi3vvWtNS1hV199NX79139dvATRw3TDxxrxD//wD45BF0/72c9+5hh08UN8FhJEN3CrOxScbUH42meSJCEajWLbtm04f/48FhYWoOu6r/FjfiDbdId26tlpI+8XXngBb37zm23BV69v5E00Rzt9zA3aXJvoR9zqDgVnW5RMJoMTJ07YdhVoZfyYH8g23cGPniuVSk0r2AsvvIBf/OIXNa1gV199NS699FLxEsQWwo+Pifz85z+vCbisR6VScQy6+L/f9ra3iZckiE2PW92h4IzYMMg23aGenv/xH/+xphXshRdewPnz5x1bwt7+9reLlyCIuj7G+X//7//VBFzWQOyHP/xhTcBlPS677DLxkgTR87jVHQrOiA2DbNMdAoEAfvGLX+Ab3/hGTZfkt7/9bceWsH/5L/+leBmCcCUQCOCXv/wl/v7v/74m6OLHd77znZqAyxqIveMd7xAvSxB9j9t7sGFwRhAEQRAEQXQGpzCsbnBGEARBEARBdBfapp4gCIIgCGITQcEZQRAEQRDEJoKCM4IgCIIgiE0EBWcEQRAEQRCbiP8fTJGYHHdCxwYAAAAASUVORK5CYII=\" style=\"width: 531px; height: 332.415px;\" width=\"531\" height=\"332.415\"\u003e\u003c/p\u003e\n\u003cp\u003eTable 2. Leukocyte counts in peripheral blood in children with or without XLRS.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"612\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003eControls (absolute count)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 216px;\"\u003e\n \u003cp\u003eXLRS (absolute count)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003eP value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003eNeutrophils\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e41.040 \u0026plusmn; 4.087 %\u003c/p\u003e\n \u003cp\u003e(3054.000 \u0026plusmn; 393.784 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 216px;\"\u003e\n \u003cp\u003e41.500 \u0026plusmn; 1.993 %\u003c/p\u003e\n \u003cp\u003e(3212.000 \u0026plusmn; 423.644 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e0.9219\u003c/p\u003e\n \u003cp\u003e(0.7916)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003eLymphocytes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e47.480 \u0026plusmn; 4.698 %\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(3474.000 \u0026plusmn; 325.032 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 216px;\"\u003e\n \u003cp\u003e47.440 \u0026plusmn; 2.946 %\u003c/p\u003e\n \u003cp\u003e(3668.000 \u0026plusmn; 489.504 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e0.9944 (0.7498)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003eMonocytes\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e8.040 \u0026plusmn; 0.995 % \u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(590.000 \u0026plusmn; 71.972 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 216px;\"\u003e\n \u003cp\u003e6.820 \u0026plusmn; 0.801 %\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(514.000 \u0026plusmn; 75.736 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e0.3677 (0.4877)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003eEosinophils\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e2.640 \u0026plusmn; 0.427 %\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(194.000 \u0026plusmn; 31.559 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 216px;\"\u003e\n \u003cp\u003e3.520 \u0026plusmn; 1.059 %\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(242.000 \u0026plusmn; 49.132 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e0.4631 (0.4349)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003eBasophils\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 211px;\"\u003e\n \u003cp\u003e0.580 \u0026plusmn; 0.058 %\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(42.000 \u0026plusmn; 3.742 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 216px;\"\u003e\n \u003cp\u003e0.540 \u0026plusmn; 0.154 %\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(38.000 \u0026plusmn; 5.831 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e0.8138 (0.5796)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eTable 3. Lymphocyte subsets in children with or without XLRS.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"637\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003eMarkers\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 214px;\"\u003e\n \u003cp\u003e% lymphocyte gate in controls (absolute count)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e% lymphocyte gate in XLRS (absolute count)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003eP value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003eCD11c+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 214px;\"\u003e\n \u003cp\u003e11.158 \u0026plusmn; 1.992 %\u003c/p\u003e\n \u003cp\u003e(400.115 \u0026plusmn; 101.864 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e7.872 \u0026plusmn; 1.035 %\u003c/p\u003e\n \u003cp\u003e(274.099 \u0026plusmn; 34.559 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e0.1814\u003c/p\u003e\n \u003cp\u003e(0.2751)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003eCD11c+/CD56+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 214px;\"\u003e\n \u003cp\u003e6.766 \u0026plusmn; 1.956 %\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(240.404 \u0026plusmn; 86.691 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e4.058 \u0026plusmn; 0.562 %\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(141.126 \u0026plusmn; 19.633 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e0.2200 (0.2965)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003eCD11c+/CD56-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 214px;\"\u003e\n \u003cp\u003e4.392 \u0026plusmn; 0.724 %\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(159.710 \u0026plusmn; 32.782 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e3.814 \u0026plusmn; 0.825 %\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(132.973 \u0026plusmn; 28.941 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e0.6128 (0.5579)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003eCD16a+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 214px;\"\u003e\n \u003cp\u003e13.248 \u0026plusmn; 3.299 %\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(467.071 \u0026plusmn; 147.728 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e4.520 \u0026plusmn; 1.274 %\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(150.752 \u0026plusmn; 43.493 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e0.0388 (0.0740)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003eCD16a+/CD56+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 214px;\"\u003e\n \u003cp\u003e10.318 \u0026plusmn; 2.741 %\u003c/p\u003e\n \u003cp\u003e(358.035 \u0026plusmn; 118.271 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e3.368 \u0026plusmn; 1.050 %\u003c/p\u003e\n \u003cp\u003e(112.628 \u0026plusmn; 36.891 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e0.0454\u003c/p\u003e\n \u003cp\u003e(0.0829)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003eCD19+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 214px;\"\u003e\n \u003cp\u003e15.200 \u0026plusmn; 2.223 %\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(544.000 \u0026plusmn; 100.923 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e20.800 \u0026plusmn; 3.121 %\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(768.600 \u0026plusmn; 142.742 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e0.182 (0.2348)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003eCD3+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 214px;\"\u003e\n \u003cp\u003e72.000 \u0026plusmn; 3.332 %\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(2481.600 \u0026plusmn; 219.593 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e72.400 \u0026plusmn; 2.977 %\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(2665.000 \u0026plusmn; 397.288 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e0.9309 (0.6968)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003eCD3+/CD4+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 214px;\"\u003e\n \u003cp\u003e36.600 \u0026plusmn; 3.140 %\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(1279.000 \u0026plusmn; 176.070 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e46.200 \u0026plusmn; 2.035 %\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(1723.800 \u0026plusmn; 276.160 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e0.0333 (0.2115)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003eCD3+/CD8+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 214px;\"\u003e\n \u003cp\u003e29.200 \u0026plusmn; 2.035 %\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(996.400 \u0026plusmn; 72.873 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e20.800 \u0026plusmn; 1.281 %\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(778.000 \u0026plusmn; 132.950 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e0.0082 (0.1877)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003eCD3+/CD4-/CD8-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 214px;\"\u003e\n \u003cp\u003e6.400 \u0026plusmn; 1.122 %\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(212.000 \u0026plusmn; 25.022 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e5.400 \u0026plusmn; 1.913 %\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(163.400 \u0026plusmn; 21.860 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e0.6641 (0.1817)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003eCD3-/CD56+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 214px;\"\u003e\n \u003cp\u003e10.600 \u0026plusmn; 2.731 %\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(369.800 \u0026plusmn; 120.221 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e5.000 \u0026plusmn; 0.707 %\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(176.400 \u0026plusmn; 28.151 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e0.0824 (0.1559)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003eCD4+/CD25+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 214px;\"\u003e\n \u003cp\u003e7.800 \u0026plusmn; 0.735 %\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(264.200 \u0026plusmn; 18.301 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e9.200 \u0026plusmn; 0.917 %\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(327.000 \u0026plusmn; 44.714 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e0.2675 (0.2299)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 143px;\"\u003e\n \u003cp\u003eCD4+/CD25+/CD127-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 214px;\"\u003e\n \u003cp\u003e3.200 \u0026plusmn; 0.374 %\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(108.000 \u0026plusmn; 10.330 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 208px;\"\u003e\n \u003cp\u003e4.200 \u0026plusmn; 0.490 %\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(159.200 \u0026plusmn; 30.871 cells/\u0026micro;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e0.1434 (0.1544)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-immunology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"imno","sideBox":"Learn more about [BMC Immunology](http://bmcimmunol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/imno/default.aspx","title":"BMC Immunology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"X-linked retinoschisis, retinoschisin, flow cytometry, CD4/CD8 ratio.","lastPublishedDoi":"10.21203/rs.3.rs-6263927/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6263927/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground.\u003c/h2\u003e \u003cp\u003eX-linked retinoschisis is a retinovitreal disorder primarily affecting males, starting in childhood. Over time, patients experience deterioration of vision due to the lack of retinoschisin-1 function. In clinical trials performing ocular gene delivery in those affected by this disorder, ocular inflammation was observed, which may have masked efficacy. A subsequent study focusing on analyzing the populations of peripheral blood mononuclear cells and cytokines in adults with this disease reported aberrant dendritic cell numbers and cytokine levels in peripheral blood. Adults with this disease may have an altered baseline immunity. However, children with X-linked retinoschisis were not included in that study, and whether the aberrant peripheral immunity in affected adults was a consequence of advanced eye pathology remained unclear. This study focuses on analyzing the populations of blood lymphocyte subsets in children aged 0 to 7 with X-linked retinoschisis and age-matched controls using flow cytometry.\u003c/p\u003e\u003ch2\u003eResults.\u003c/h2\u003e \u003cp\u003eThe fractions of lymphocyte subsets that were CD16a\u0026thinsp;+\u0026thinsp;were significantly lower in children with X-linked retinoschisis. The fractions of lymphocyte subsets that were CD16a+/CD56+, namely natural killer cells, were also significantly lower. In children with X-linked retinoschisis, the fractions of CD3+/CD4\u0026thinsp;+\u0026thinsp;T cells were higher, and the fractions of CD3\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;T cells were lower, despite having the same amounts of total CD3\u0026thinsp;+\u0026thinsp;T cells within their lymphocyte populations. This resulted in a significantly greater CD4/CD8 ratio in children with X-linked retinoschisis compared to age-matched controls.\u003c/p\u003e\u003ch2\u003eConclusions.\u003c/h2\u003e \u003cp\u003eAlterations were found in blood lymphocyte compositions of children with X-linked retinoschisis within both innate and adaptive immune axes. Some alterations including an elevation of CD4/CD8 ratio in X-linked retinoschisis mirror those previously found in adult patients with this disease. The fact that these abnormalities were present early in this disease indicates that retinoschisin-1 may play a role in regulating immunity in addition to retinal adhesion. The findings may have implications for future treatments such as ocular gene delivery.\u003c/p\u003e","manuscriptTitle":"Immune landscape in children with X-linked retinoschisis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-17 10:42:39","doi":"10.21203/rs.3.rs-6263927/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-22T15:15:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-22T09:16:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-15T14:38:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"59115404571437452548919690771221945357","date":"2025-04-07T20:31:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"58213316121375014773082971711602660945","date":"2025-04-07T17:22:07+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-28T16:39:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-28T15:56:55+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-03-26T06:40:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-25T19:10:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Immunology","date":"2025-03-25T19:09:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-immunology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"imno","sideBox":"Learn more about [BMC Immunology](http://bmcimmunol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/imno/default.aspx","title":"BMC Immunology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a48752a8-ba99-4840-b53b-eb071f043d22","owner":[],"postedDate":"April 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-07-11T08:38:45+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-17 10:42:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6263927","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6263927","identity":"rs-6263927","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}