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Deep dermal and full-thickness burns often result in scar sequelae such as contractures, hypertrophy, pain and itching following split-thickness skin grafting. Dermal substitutes are currently employed alongside split-thickness skin grafting to enhance clinical outcomes, though their indications remain a subject of ongoing debate. This systematic review aims to clarify the indications for the application of dermal substitutes in burn patients, in both acute and reconstructive settings. A comprehensive search across various databases was conducted. Studies (n = 190) assessing the indications and outcomes of dermal substitutes in acute burn patients and those requiring reconstructive surgery were included. Data extraction included the applied dermal substitute, age, total body surface area, wound depth, burn aetiology, anatomical site and exclusion criteria. The indications were derived from predetermined indications, i.e. inclusion and exclusion criteria and patient characteristics. The depth of the wound emerged as the primary indication for dermal substitute use. A one-stage approach is recommended for deep dermal to full-thickness wounds larger than 10 cm 2 , while a two-stage approach is advised for wounds of this depth with limited donor sites or exposed bone or tendon. No definitive age or burn/scar location thresholds were identified, and careful consideration is advised for electrical and chemical burns. Contraindications include wound infections and allergies to matrix components. Limited data exist on use in patients with diabetes mellitus, chronic vascular disease, or immunocompromised status. This is the first review to address the indications for dermal substitutes in burn patients, providing valuable insights for the development of international evidence-based treatment guidelines., (© 2024 The Author(s). Wound Repair and Regeneration published by Wiley Periodicals LLC on behalf of The Wound Healing Society.)

Dermal substitutes have been introduced in burn care to improve wound healing outcomes; however, their use remains limited in standard treatments. This systematic review and meta-analysis aimed to evaluate the outcomes of dermal substitutes in patients with burns and patients requiring burn scar reconstruction and subsequently contribute to optimising the integration of dermal substitutes into clinical practice and reducing the knowledge gap. A comprehensive search across various databases included human studies from peer-reviewed journals on dermal substitutes for deep dermal and full-thickness burns, and scar reconstruction across all ages. Data from comparative trials were extracted, focusing on patient and wound characteristics, treatment specifics, and outcomes related to wound healing and scar quality. Meta-analysis was performed on trials reporting similar post-burn measures, with statistical heterogeneity assessed. Outcomes were presented using mean differences or odds ratios with 95% confidence intervals. A total of 31 comparative trials were included. The overall quality of the studies was considered moderate. The meta-analysis indicated delayed re-epithelialization 4-7 days after treatment with a collagen-elastin matrix compared to split-thickness skin graft in acute burns (-7.30%, p = 0.02). Significant improvement in subjective scar quality was observed with acellular dermal matrix compared to split-thickness skin graft in acute burn wounds 6 months post-operative (-1.95, p <0.01). While acknowledging the initially delayed wound healing, incorporating dermal substitutes into the surgical treatment of burn patients holds promise for enhancing scar quality. However, future research must prioritise outcome measure uniformity, address variations in dermal substitute application, and standardise indications for consistent and effective practices., (© 2024 The Author(s). Wound Repair and Regeneration published by Wiley Periodicals LLC on behalf of The Wound Healing Society.)

Background: Value-based healthcare (VBHC) is increasingly implemented in healthcare worldwide. Transparent measurement of the outcomes most important and relevant to patients is essential in VBHC, which is supported by a core set of most important quality indicators and outcomes. Therefore, the aim of this study was to develop a VBHC-burns core set for adult burn patients., Methods: A three-round modified national Delphi study, including 44 outcomes and 24 quality indicators, was conducted to reach consensus among Dutch patients, burn care professionals and researchers. Items were rated on a nine-point Likert scale and selected if ≥ 70% in each group considered an item 'important'. Subsequently, instruments quantifying selected outcomes were identified based on a literature review and were chosen in a consensus meeting using recommendations from the Dutch consensus-based standard set and the Dutch Centre of Expertise on Health Disparities. Time assessment points were chosen to reflect the burn care and patient recovery process. Finally, the initial core set was evaluated in practice, leading to the adapted VBHC-burns core set., Results: Twenty-seven patients, 63 burn care professionals and 23 researchers participated. Ten outcomes and four quality indicators were selected in the Delphi study, including the outcomes pain, wound healing, physical activity, self-care, independence, return to work, depression, itching, scar flexibility and return to school. Quality indicators included shared decision-making (SDM), the number of patients receiving aftercare, determination of burn depth, and assessment of active range of motion. After evaluation of its use in clinical practice, the core set included all items except SDM, which are assessed by 9 patient-reported outcome instruments or measured in clinical care. Assessment time points included are at discharge, 2 weeks, 3 months, 12 months after discharge and annually afterwards., Conclusion: A VBHC-burns core set was developed, consisting of outcomes and quality indicators that are important to burn patients and burn care professionals. The VBHC-burns core set is now systemically monitored and analysed in Dutch burn care to improve care and patient relevant outcomes. As improving burn care and patient relevant outcomes is important worldwide, the developed VBHC-burns core set could be inspiring for other countries., Competing Interests: Declaration of Competing Interest Not applicable., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Aims: Aging is a dominant driver of atherosclerosis and induces a series of immunological alterations, called immunosenescence. Given the demographic shift towards elderly, elucidating the unknown impact of aging on the immunological landscape in atherosclerosis is highly relevant. While the young Western diet-fed Ldlr-deficient (Ldlr-/-) mouse is a widely used model to study atherosclerosis, it does not reflect the gradual plaque progression in the context of an aging immune system as occurs in humans., Methods and Results: Here, we show that aging promotes advanced atherosclerosis in chow diet-fed Ldlr-/- mice, with increased incidence of calcification and cholesterol crystals. We observed systemic immunosenescence, including myeloid skewing and T-cells with more extreme effector phenotypes. Using a combination of single-cell RNA-sequencing and flow cytometry on aortic leucocytes of young vs. aged Ldlr-/- mice, we show age-related shifts in expression of genes involved in atherogenic processes, such as cellular activation and cytokine production. We identified age-associated cells with pro-inflammatory features, including GzmK+CD8+ T-cells and previously in atherosclerosis undefined CD11b+CD11c+T-bet+ age-associated B-cells (ABCs). ABCs of Ldlr-/- mice showed high expression of genes involved in plasma cell differentiation, co-stimulation, and antigen presentation. In vitro studies supported that ABCs are highly potent antigen-presenting cells. In cardiovascular disease patients, we confirmed the presence of these age-associated T- and B-cells in atherosclerotic plaques and blood., Conclusions: Collectively, we are the first to provide comprehensive profiling of aged immunity in atherosclerotic mice and reveal the emergence of age-associated T- and B-cells in the atherosclerotic aorta. Further research into age-associated immunity may contribute to novel diagnostic and therapeutic tools to combat cardiovascular disease., Competing Interests: Conflict of interest: None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Background and Aims: Scavenger receptor class B1 (SCARB1) - also known as the high-density lipoprotein (HDL) receptor - is a multi-ligand scavenger receptor that is primarily expressed in liver and steroidogenic organs. This receptor is known for its function in reverse cholesterol transport (RCT) in mammals and hence disruption leads to a massive increase in HDL cholesterol in these species. The extracellular domain of SCARB1 - which is important for cholesterol handling - is highly conserved across multiple vertebrates, except in zebrafish., Methods: To examine the functional conservation of SCARB1 among vertebrates, two stable scarb1 knockout zebrafish lines, scarb1 715delA (scarb1 -1 nt) and scarb1 715&#95;716insGG (scarb1 +2 nt), were created using CRISPR-Cas9 technology., Results: We demonstrate that, in zebrafish, SCARB1 deficiency leads to disruption of carotenoid-based pigmentation, reduced fertility, and a decreased larvae survival rate, whereas steroidogenesis was unaltered. The observed reduced fertility is driven by defects in female fertility (-50 %, p < 0.001). Importantly, these alterations were independent of changes in free (wild-type 2.4 ± 0.2 μg/μl versus scarb1 -/- 2.0 ± 0.1 μg/μl) as well as total (wild-type 4.2 ± 0.4 μg/μl versus scarb1 -/- 4.0 ± 0.3 μg/μl) plasma cholesterol levels. Uptake of HDL in the liver of scarb1 -/- zebrafish larvae was reduced (-86.7 %, p < 0.001), but this coincided with reduced perfusion of the liver. No effect was observed on lipoprotein uptake in the caudal vein. SCARB1 deficient canaries, which also lack carotenoids in their plumage, similarly as scarb1 -/- zebrafish, failed to show an increase in plasma free- and total cholesterol levels., Conclusion: Our findings suggest that the specific function of SCARB1 in maintaining plasma cholesterol could be an evolutionary novelty that became prominent in mammals, while other known functions were already present earlier during vertebrate evolution., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Protein arginine methyltransferase 5 (PRMT5) controls inflammation and metabolism through modulation of histone methylation and gene transcription. Given the important role of inflammation and metabolism in atherosclerotic cardiovascular disease, here we examined the role of PRMT5 in atherosclerosis using the specific PRMT5 inhibitor GSK3326595. Cultured thioglycollate-elicited peritoneal macrophages were exposed to GSK3326595 or DMSO control and stimulated with either 1 ng/mL LPS or 100 ng/mL interferon-gamma for 24 h. Furthermore, male low-density lipoprotein (LDL) receptor knockout mice were fed an atherogenic Western-type diet and injected intraperitoneally 3×/week with a low dose of 5 mg/kg GSK3326595 or solvent control for 9 weeks. In vitro, GSK3326595 primed peritoneal macrophages to interferon-gamma-induced M1 polarization, as evidenced by an increased M1/M2 gene marker ratio. In contrast, no difference was found in the protein expression of iNOS (M1 marker) and ARG1 (M2 marker) in peritoneal macrophages of GSK3326595-treated mice. Also no change in the T cell activation state or the susceptibility to atherosclerosis was detected. However, chronic GSK3326595 treatment did activate genes involved in hepatic fatty acid acquisition, i.e. SREBF1, FASN, and CD36 (+59%, +124%, and +67%, respectively; p < 0.05) and significantly increased hepatic triglyceride levels (+50%; p < 0.05). PRMT5 inhibition by low-dose GSK3326595 treatment does not affect the inflammatory state or atherosclerosis susceptibility of Western-type diet-fed LDL receptor knockout mice, while it induces hepatic triglyceride accumulation. Severe side effects in liver, i.e. development of non-alcoholic fatty liver disease, should thus be taken into account upon chronic treatment with this PRMT5 inhibitor., (© 2023 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background and Aims: Scavenger receptors form a superfamily of membrane-bound receptors that bind and internalize different types of ligands, including pro-atherogenic oxidized low-density lipoproteins (oxLDLs). In vitro studies have indicated a role for the liver sinusoidal endothelial cell receptors stabilin 1 (stab1) and 2 (stab2) in oxLDL clearance. In this study, we evaluated the potential role of stab1 and stab2 in lipoprotein uptake in zebrafish, an upcoming model for studying cholesterol metabolism and atherosclerosis., Methods: Lipoproteins were injected in the duct of Cuvier of wild-type (ABTL) or stab1 and stab2 mutant (stab1 -/- stab2 -/- ) zebrafish larvae at 3 days post-fertilization. To examine the effect of stabilin deficiency on lipoprotein and cholesterol metabolism, zebrafish larvae were challenged with a high cholesterol diet (HCD; 4% w/w) for 10 days., Results: Lipoprotein injections showed impaired uptake of both LDL and oxLDL into the vessel wall of caudal veins of stab1 -/- stab2 -/- zebrafish, which was paralleled by redistribution to tissue macrophages. Total body cholesterol levels did not differ between HCD-fed stab1 -/- stab2 -/- and ABTL zebrafish. However, stab1 -/- stab2 -/- larvae exhibited 1.4-fold higher mRNA expression levels of ldlra involved in (modified) LDL uptake, whereas the expression levels of scavenger receptors scarb1 and cd36 were significantly decreased., Conclusions: We have shown that stabilins 1 and 2 have an important scavenging function for apolipoprotein B-containing lipoproteins in zebrafish and that combined deficiency of these two proteins strongly upregulates the clearance of lipoproteins by macrophages within the caudal vein. Our current study highlights the use of zebrafish as model to study lipoprotein metabolism and liver sinusoidal endothelial cell function., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background and Aims: Atherosclerotic cardiovascular disease is a metabolic and inflammatory disorder. In vitro studies have suggested that protein arginine methyltransferase 4 (PRMT4) may act as a transcriptional coactivator to modulate inflammatory and metabolic processes. Here we investigated the potential anti-atherogenic effect of PRMT4 inhibitor TP-064 in vivo., Methods: Male apolipoprotein E knockout mice fed a high cholesterol/high fat Western-type diet were intraperitoneally injected three times a week with 2.5 mg/kg (low dose) or 10 mg/kg (high dose) TP-064 or with DMSO control., Results: TP-064 induced a dose-dependent decrease in lipopolysaccharide-induced ex vivo blood monocyte Tnfα secretion (p < 0.05 for trend) in the context of unchanged blood monocyte concentrations and neutrophilia induction (p < 0.01 for trend). A dose-dependent decrease in gonadal white adipose tissue expression levels of PPARγ target genes was detected, which translated into a reduced body weight gain after high dose TP-064 treatment (p < 0.05). TP-064 treatment also dose-dependently downregulated gene expression of the glycogen metabolism related protein G6pc in the liver (p < 0.001 for trend). In addition, a trend towards lower plasma insulin and higher blood glucose levels was observed, which was paralleled by a reduction in hepatic mRNA expression levels of the insulin-responsive genes Fasn (-55%; p < 0.001) and Gck (-47%; p < 0.001) in high dose-treated mice. Plasma triglyceride levels were reduced by high dose TP-064 treatment (-30%; p < 0.05). However, no change was observed in the size or composition of aortic root atherosclerotic lesions., Conclusions: The PRMT4 inhibitor TP-064 impacts both inflammatory and metabolic processes without changing atherosclerosis susceptibility of male apolipoprotein E knockout mice., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Aim: Signaling through the coinhibitory programmed death (PD)-1/PD-L1 pathway regulates T cell responses and can inhibit ongoing immune responses. Inflammation is a key process in the development of atherosclerosis, the underlying cause for the majority of cardiovascular diseases. Dampening the excessive immune response that occurs during atherosclerosis progression by promoting PD-1/PD-L1 signaling may have a high therapeutic potential to limit disease burden. In this study we therefore aimed to assess whether an agonistic PD-1 antibody can diminish atherosclerosis development. Methods and Results: Ldlr -/- mice were fed a western-type diet (WTD) while receiving 100 μg of an agonistic PD-1 antibody or control vehicle twice a week. Stimulation of the PD-1 pathway delayed the WTD-induced monocyte increase in the circulation up to 3 weeks and reduced T cell activation and proliferation. CD4 + T cell numbers in the atherosclerotic plaque were reduced upon PD-1 treatment. More specifically, we observed a 23% decrease in atherogenic IFNγ-producing splenic CD4 + T cells and a 20% decrease in cytotoxic CD8 + T cells, whereas atheroprotective IL-10 producing CD4 + T cells were increased with 47%. Furthermore, we found an increase in regulatory B cells, B1 cells and associated atheroprotective circulating oxLDL-specific IgM levels in agonistic PD-1-treated mice. This dampened immune activation following agonistic PD-1 treatment resulted in reduced atherosclerosis development ( p < 0.05). Conclusions: Our data show that stimulation of the coinhibitory PD-1 pathway inhibits atherosclerosis development by modulation of T- and B cell responses. These data support stimulation of coinhibitory pathways as a potential therapeutic strategy to combat atherosclerosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Grievink, Smit, Verwilligen, Bernabé Kleijn, Smeets, Binder, Yagita, Moerland, Kuiper, Bot and Foks.)

CD47, also known as integrin-associated protein (IAP), is a transmembrane protein with multiple biological functions including regulation of efferocytosis and leukocyte trafficking. In this study we investigated the effect of CD47-deficiency on atherosclerosis using a model of adeno-associated virus (AAV)-induced hypercholesterolemia. We observed increased plaque formation in CD47 null mice compared to wild-type controls. Loss of CD47 caused activation of dendritic cells, T cells and natural killer (NK) cells, indicating an important role for CD47 in regulating immunity. In particular, Cd47 deficiency increased the proportion of IFN-γ producing CD90 + NK cells. Treatment with depleting anti-NK1.1 monoclonal antibody (mAb), but not depleting anti-CD4/CD8 mAbs, equalized atherosclerotic burden, suggesting NK cells were involved in the enhanced disease in Cd47 deficient mice. Additional studies revealed that levels of CD90 + and IFN-γ + NK cells were expanded in atherosclerotic aorta and that CD90 + NK cells produce more IFN-γ than CD90 - NK cells. Finally, we demonstrate that anti-CD47 (MIAP410) causes splenomegaly and activation of DCs and T cells, without affecting NK cell activation. In summary, we demonstrate that loss of CD47 causes increased lymphocyte activation that results in increased atherosclerosis.

Apolipoprotein E (APOE) deficient mice exhibit unexplained hypercorticosteronemia. Given that APOE is also produced locally within the adrenals, we evaluated the effect of adrenal-specific APOE deficiency on the glucocorticoid function. Hereto, one adrenal containing or lacking APOE was transplanted into adrenalectomized wild-type mice. Adrenal APOE deficiency did not impact adrenal total cholesterol levels. Importantly, the ability of the two adrenal types to produce glucocorticoids was also not different as judged from the similar plasma corticosterone levels. Adrenal mRNA expression levels of HMG-CoA reductase and the LDL receptor were decreased by respectively 72% (p < 0.01) and 65% (p = 0.07), suggesting that cholesterol acquisition pathways were inhibited to possibly compensate the lack of APOE. In support, a parallel increase in the expression level of the cholesterol accumulation-associated ER stress marker CHOP was detected (+117%; p < 0.05). In conclusion, our studies show that elimination of adrenocortical APOE production does not impact glucocorticoid output in wild-type mice., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Atherothrombotic events such as myocardial infarction and ischemic stroke are a major cause of morbidity and mortality worldwide. Understanding the molecular and cellular mechanisms of atherosclerotic plaque destabilization or erosion, and developing new therapeutics to prevent acute cardiovascular events is important for vascular biology research and clinical cardiovascular medicine. However, basic research on plaque destabilization, rupture and erosion is hampered by the lack of appropriate animal models of atherothrombosis. Unprovoked atherothrombosis is very scarce in commonly used mouse models for atherosclerosis, the low-density lipoprotein receptor knockout and apolipoprotein E knockout mice. Therefore, specific interventions are required to induce atherothrombosis in these models. Two strategies can be employed to induce atherothrombosis: 1) plaque destabilization and 2) induction of blood hypercoagulability. Although the individual strategies yield atherothrombosis at low incidence, it appears that the combination of both plaque destabilization and an increase in blood coagulability is the most promising strategy to induce atherothrombosis on a larger scale. In this review, we summarize the recent developments on mouse models for the investigation of atherothrombosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background and Aims: Although studies in mice have suggested that lesion regression is feasible, the underlying mechanisms remain largely unknown. Here we determined the impact of high-density lipoprotein (HDL) on atherosclerosis regression outcome., Methods: Atherosclerotic lesion dynamics were studied upon bone marrow transplantation-mediated re-introduction of apolipoprotein E (Apoe) in Apoe knockout mice. Probucol was used to pharmacologically deplete HDL., Results: Restoration of Apoe function was associated with an initial growth of atherosclerotic lesions and parallel decrease in lesional macrophage foam cell content (47 ± 4% at 4 weeks versus 72 ± 2% at baseline: p < 0.001), despite the fact that cholesterol levels were markedly reduced. Notably, significant lesion regression was detected from 4 weeks onwards, when plasma cholesterol levels had returned to the normolipidemic range. As a result, lesions were 41% smaller (p < 0.05) at 8 weeks than at 4 weeks after bone marrow transplantation. Regressed lesions contained an even lower level of macrophage foam cells (33 ± 5%: p < 0.001) and were rich in collagen. Probucol co-treatment was associated with a 3.2-fold lower (p < 0.05) plasma HDL-cholesterol level and a more pro-inflammatory (CCR2+) monocyte phenotype. Importantly, probucol-treated mice exhibited atherosclerotic lesions that were larger than those of regular chow diet-fed bone marrow transplanted mice at 8 weeks (186 ± 15*10 3  μm 2 for probucol-treated versus 120 ± 19*10 3  μm 2 for controls: p < 0.05)., Conclusions: We have shown that probucol-induced HDL deficiency impairs the ability of established lesions to regress in response to reversal of the genetic hypercholesterolemia in Apoe knockout mice. Our studies thus highlight a crucial role for HDL in the process of atherosclerosis regression., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Interleukin-23 (IL-23) has been implicated in inflammatory and autoimmune diseases by skewing CD4 + T helper cells towards a pathogenic Th17 phenotype. In this study we investigated the presence of IL-23 receptor (IL-23R)-expressing cells in the atherosclerotic aorta and evaluated the effect of IL-23R deficiency on atherosclerosis development in mice., Methods and Results: We used heterozygous Ldlr -/- Il23r e GFP / WT knock-in mice to identify IL-23R-expressing cells by flow cytometry and homozygous Ldlr -/- Il23r e GFP / eGFP ( Ldlr -/- Il23r -/- ) mice to investigate the effect of lack of IL-23R in atherosclerosis. We demonstrate the presence of relatively rare IL-23R-expressing cells in lymphoid tissue and aorta (≈0.1-1% IL23R + cells of all CD45 + leukocytes). After 10 weeks on a high-fat diet, production of IL-17, but not interferon-γ, by CD4 + T cells and other lymphocytes was reduced in Ldlr -/- Il23r -/- compared with Ldlr -/- controls. However, Ldlr -/- and Ldlr -/- Il23r -/- mice had equivalent amounts of aortic sinus and descending aorta lesions. Adoptive transfer of IL-23R-deficient CD4 + T cells to lymphopenic Ldlr -/- Rag1 -/- resulted in dramatically reduced IL-17-producing T cells but did not reduce atherosclerosis, compared with transfer of IL-23R-sufficient CD4 + T cells., Conclusions: These data demonstrate that loss of IL-23R does not affect development of experimental atherosclerosis in LDLr-deficient mice, despite a role for IL-23 in differentiation of IL-17-producing T cells., (© 2018 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.)

Objective: Murine atherosclerosis models do not spontaneously develop atherothrombotic complications. We investigated whether disruption of natural anticoagulation allows preexisting atherosclerotic plaques to progress toward an atherothrombotic phenotype., Approach and Results: On lowering of plasma protein C levels with small interfering RNA (si Proc ) in 8-week Western-type diet-fed atherosclerotic apolipoprotein E-deficient mice, 1 out of 4 mice displayed a large, organized, and fibrin- and leukocyte-rich thrombus on top of an advanced atherosclerotic plaque located in the aortic root. Although again at low incidence (3 in 25), comparable thrombi at the same location were observed during a second independent experiment in 9-week Western-type diet-fed apolipoprotein E-deficient mice. Mice with thrombi on their atherosclerotic plaques did not show other abnormalities and had equally lowered plasma protein C levels as si Proc -treated apolipoprotein E-deficient mice without thrombi. Fibrinogen and thrombin-antithrombin concentrations and blood platelet numbers were also comparable, and plaques in si Proc mice with thrombi had a similar composition and size as plaques in si Proc mice without thrombi. Seven out of 25 si Proc mice featured clots in the left atrium of the heart., Conclusions: Our findings indicate that small interfering RNA-mediated silencing of protein C in apolipoprotein E-deficient mice creates a condition that allows the occurrence of spontaneous atherothrombosis, albeit at a low incidence. Lowering natural anticoagulation in atherosclerosis models may help to discover factors that increase atherothrombotic complications., (© 2017 American Heart Association, Inc.)