Engineering immune-evasive allogeneic cellular immunotherapies.

Allogeneic cellular immunotherapies hold a great promise for cancer treatment owing to their potential cost-effectiveness, scalability and on-demand availability. However, immune rejection of adoptively transferred allogeneic T and natural killer (NK) cells is a substantial obstacle to achieving clinical responses that are comparable to responses obtained with current autologous chimeric antigen receptor T cell therapies. In this Perspective, we discuss strategies to confer cell-intrinsic, immune-evasive properties to allogeneic T cells and NK cells in order to prevent or delay their immune rejection, thereby widening the therapeutic window. We discuss how common viral and cancer immune escape mechanisms can serve as a blueprint for improving the persistence of off-the-shelf allogeneic cell therapies. The prospects of harnessing genome editing and synthetic biology to design cell-based precision immunotherapies extend beyond programming target specificities and require careful consideration of innate and adaptive responses in the recipient that may curtail the biodistribution, in vivo expansion and persistence of cellular therapeutics.

Genetic ablation of adhesion ligands averts rejection of allogeneic immune cells.

Allogeneic cell therapies hold promise for broad clinical implementation, but face limitations due to potential rejection by the recipient immune system. Silencing of beta-2-microglobulin ( B2M ) expression is commonly employed to evade T cell-mediated rejection, although absence of B2M triggers missing-self responses by recipient natural killer (NK) cells. Here, we demonstrate that deletion of the adhesion ligands CD54 and CD58 on targets cells robustly dampens NK cell reactivity across all sub-populations. Genetic deletion of CD54 and CD58 in B2M -deficient allogeneic chimeric antigen receptor (CAR) T and multi-edited induced pluripotent stem cell (iPSC)-derived NK cells reduces their susceptibility to rejection by NK cells in vitro and in vivo without affecting their anti-tumor effector potential. Thus, these data suggest that genetic ablation of adhesion ligands effectively alleviates rejection of allogeneic immune cells for immunotherapy.

Delayed graft rejection in autoimmune islet transplantation via biomaterial immunotherapy.

The induction of operational immune tolerance is a major goal in beta-cell replacement strategies for the treatment of type 1 diabetes. Our group previously reported long-term efficacy via biomaterial-mediated programmed death ligand 1 (PD-L1) immunotherapy in islet allografts in nonautoimmune models. In this study, we evaluated autoimmune recurrence and allograft rejection during islet transplantation in spontaneous nonobese diabetic (NOD) mice. Graft survival and metabolic function were significantly prolonged over 60 days in recipients of syngeneic islets receiving the biomaterial-delivered immunotherapy, but not in control animals. The biomaterial-mediated PD-L1 immunotherapy resulted in delayed allograft rejection in diabetic NOD mice compared with controls. Discrimination between responders and nonresponders was attributed to the enriched presence of CD206+ program death 1+ macrophages and exhausted signatures in the cytotoxic T cell compartment in the local graft microenvironment. Notably, draining lymph nodes had similar remodeling in innate and adaptive immune cell populations. This work establishes that our biomaterial platform for PD-L1 delivery can modulate immune responses to transplanted islets in diabetic NOD mice and, thus, can provide a platform for the development of immunologic strategies to curb the allo- and autoimmune processes in beta-cell transplant recipients.

Hydrolytic hydrogels tune mesenchymal stem cell persistence and immunomodulation for enhanced diabetic cutaneous wound healing.

Diabetes is associated with an altered global inflammatory state with impaired wound healing. Mesenchymal stem/stromal cells (MSC) are being explored for treatment of diabetic cutaneous wounds due to their regenerative properties. These cells are commonly delivered by injection, but the need to prolong the retention of MSC at sites of injury has spurred the development of biomaterial-based MSC delivery vehicles. However, controlling biomaterial degradation rates in vivo remains a therapeutic-limiting challenge. Here, we utilize hydrolytically degradable ester linkages to engineer synthetic hydrogels with tunable in vivo degradation kinetics for temporally controlled delivery of MSC. In vivo hydrogel degradation rate can be controlled by altering the ratio of ester to amide linkages in the hydrogel macromers. These hydrolytic hydrogels degrade at rates that enable unencumbered cutaneous wound healing, while enhancing the local persistence MSC compared to widely used protease-degradable hydrogels. Furthermore, hydrogel-based delivery of MSC modulates local immune responses and enhances cutaneous wound repair in diabetic mice. This study introduces a simple strategy for engineering tunable degradation modalities into synthetic biomaterials, overcoming a key barrier to their use as cell delivery vehicles.

Measuring for primary prevention: An online survey of local community perspectives on family and domestic violence in regional Australia.

BACKGROUND: Family and domestic violence, encompassing diverse behaviours including physical, sexual, emotional and financial abuse, is endemic worldwide and has multiple adverse health and social consequences. Principal drivers include traditional gender values that disempower women. Changing these is a key prevention strategy. In Australia, high-quality national surveys provide data on public perspectives concerning family and domestic violence but may not capture community-level diversity. As part of a project for primary prevention family and domestic violence in outer regional Australia, our aims were to develop and administer a questionnaire-based survey suitable for the local community encompassing knowledge about, attitudes towards, and personal experiences of family and domestic violence, to describe and to investigate the theoretical (factor) structure and local socio-demographic predictors of responses, and to determine the extent to which the survey findings are locally distinctive. METHODS: The online community survey for local residents (≥15 years), comprised items on respondents' sociodemographic characteristics plus questions abridged from pre-existing national instruments on knowledge about, attitudes towards, and personal experiences of family and domestic violence. Responses were rake-weighted to correct census-ascertained sample imbalance and investigated using exploratory factor analysis, with sociodemographic predictors determined using multiple linear regression and dominance analysis. RESULTS: Among 914 respondents, males (27.0%), those from age-group extremes, and less-educated persons were underrepresented. Familiarity with diverse family and domestic violence behaviours was high among all subgroups. Poorer knowledge of the FDV behaviour continuum and attitudes supporting traditional gender roles and FDV were disproportionately evident among males, older respondents and those with lower education levels. Both the factor structure of extracted composite measures reflecting community perspectives and sociodemographic predictors of responses generally aligned with patterns evident in national data. CONCLUSIONS: Local reinforcement of existing nationwide findings on community understanding of and attitudes towards family and domestic violence provides salience for targeted interventions.

Combinatorial treatment rescues tumour-microenvironment-mediated attenuation of MALT1 inhibitors in B-cell lymphomas.

Activated B-cell-like diffuse large B-cell lymphomas (ABC-DLBCLs) are characterized by constitutive activation of nuclear factor κB driven by the B-cell receptor (BCR) and Toll-like receptor (TLR) pathways. However, BCR-pathway-targeted therapies have limited impact on DLBCLs. Here we used >1,100 DLBCL patient samples to determine immune and extracellular matrix cues in the lymphoid tumour microenvironment (Ly-TME) and built representative synthetic-hydrogel-based B-cell-lymphoma organoids accordingly. We demonstrate that Ly-TME cellular and biophysical factors amplify the BCR-MYD88-TLR9 multiprotein supercomplex and induce cooperative signalling pathways in ABC-DLBCL cells, which reduce the efficacy of compounds targeting the BCR pathway members Bruton tyrosine kinase and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1). Combinatorial inhibition of multiple aberrant signalling pathways induced higher antitumour efficacy in lymphoid organoids and implanted ABC-DLBCL patient tumours in vivo. Our studies define the complex crosstalk between malignant ABC-DLBCL cells and Ly-TME, and provide rational combinatorial therapies that rescue Ly-TME-mediated attenuation of treatment response to MALT1 inhibitors.

Host type 2 immune response to xenogeneic serum components impairs biomaterial-directed osteo-regenerative therapies.

The transformative potential of cells as therapeutic agents is being realized in a wide range of applications, from regenerative medicine to cancer therapy to autoimmune disorders. The majority of these therapies require ex vivo expansion of the cellular product, often utilizing fetal bovine serum (FBS) in the culture media. However, the impact of residual FBS on immune responses to cell therapies and the resulting cell therapy outcomes remains unclear. Here, we show that hydrogel-delivered FBS elicits a robust type 2 immune response characterized by infiltration of eosinophils and CD4+ T cells. Host secretion of cytokines associated with type 2 immunity, including IL-4, IL-5, and IL-13, is also increased in FBS-containing hydrogels. We demonstrate that the immune response to xenogeneic serum components dominates the local environment and masks the immunomodulatory effects of biomaterial-delivered mesenchymal stromal/stem cells. Importantly, delivery of relatively small amounts of FBS (3.2% by volume) within BMP-2-containing biomaterial constructs dramatically reduces the ability of these constructs to promote de novo bone formation in a radial defect model in immunocompetent mice. These results urge caution when interpreting the immunological and tissue repair outcomes in immunocompetent pre-clinical models from cells and biomaterial constructs that have come in contact with xenogeneic serum components.

Hydrolytically Degradable Microgels with Tunable Mechanical Properties Modulate the Host Immune Response.

Hydrogel microparticles (microgels) are an attractive approach for therapeutic delivery because of their modularity, injectability, and enhanced integration with the host tissue. Multiple microgel fabrication strategies and chemistries have been implemented, yet manipulation of microgel degradability and its effect on in vivo tissue responses remains underexplored. Here, the authors report a facile method to synthesize microgels crosslinked with ester-containing junctions to afford tunable degradation kinetics. Monodisperse microgels of maleimide-functionalized poly(ethylene-glycol) are generated using droplet microfluidics crosslinked with thiol-terminated, ester-containing molecules. Tunable mechanics are achievable based on the ratio of degradable to nondegradable crosslinkers in the continuous phase. Degradation in an aqueous medium leads to microgel deformation based on swelling and a decrease in elastic modulus. Furthermore, degradation byproducts are cytocompatible and do not cause monocytic cell activation under noninflammatory conditions. These injectable microgels possess time-dependent degradation on the order of weeks in vivo. Lastly, the evaluation of tissue responses in a subcutaneous dorsal pocket shows a dynamic type-1 like immune response to the synthetic microgels, driven by interferon gamma (IFN-γ ) expression, which can be moderated by tuning the degradation properties. Collectively, this study demonstrates the development of a hydrolytic microgel platform that can be adapted to desired host tissue immune responses.

Towards an Evidence-Based Model of Workplace Postvention.

This instrumental case study explored what suicide postvention might offer workplaces using the example of a large metropolitan funeral company. A mixed methods approach was utilized to examine staff experiences with suicide bereavement funerals and responses to a bespoke postvention training package. Staff found funerals due to suicide difficult in terms of communication, engagement and emotionality. These challenges were commonly characterized by increased tension and concern. In the absence of a postvention informed approach, staff had developed individual ways to negotiate the identified challenges of this work. The introduction of a staff-informed postvention training package delivered improvements in staff confidence with communication, understanding and management of the impact of suicide bereavement, and increased willingness to share information about postvention services with families and mourners. The findings indicated that benefits of the training could be extended through organizational governance and integration of supports. The findings are used to inform a model of workplace postvention together with a methodology incorporating staff experience and organizational context.

High-Throughput On-Chip Human Mesenchymal Stromal Cell Potency Prediction.

Human mesenchymal stromal cells (hMSCs) are a promising source for regenerative cell therapy. However, hMSC clinical use has been stymied by product variability across hMSC donors and manufacturing practices resulting in inconsistent clinical outcomes. The inability to predict hMSC clinical efficacy, or potency, is a major limitation for market penetration. Standard metrics of hMSC potency employ hMSCs and third-party immune cell co-cultures, however, these assays face translational challenges due to third-party donor variability and lack of scalability. While surrogate markers of hMSC potency have been suggested, none have yet had translational success. To address this, a high-throughput, scalable, low-cost, on-chip microfluidic potency assay is presented with improved functional predictive power and recapitulation of in vivo secretory responses compared to traditional approaches. Comparison of hMSC secretory responses to functional hMSC-medicated immune cell suppression demonstrates shortcomings of current surrogate potency markers and identifies on-chip microfluidic potency markers with improved functional predictive power compared to traditional planar methods. Furthermore, hMSC secretory performance achieved in the on-chip microfluidic system has improved similarity compared to an in vivo model. The results underscore the shortcomings of current culture practices and present a novel system with improved functional predictive power and hMSC physiological responses.

Macrophage phenotypes in tissue repair and the foreign body response: Implications for biomaterial-based regenerative medicine strategies.

Macrophages are a highly heterogeneous and plastic population of cells that are crucial for tissue repair and regeneration. This has made macrophages a particularly attractive target for biomaterial-directed regenerative medicine strategies. However, macrophages also contribute to adverse inflammatory and fibrotic responses to implanted biomaterials, typically related to the foreign body response (FBR). The traditional model in the field asserts that the M2 macrophage phenotype is pro-regenerative and associated with positive wound healing outcomes, whereas the M1 phenotype is pro-inflammatory and associated with pathogenesis. However, recent studies indicate that both M1 and M2 macrophages play different, but equally vital, roles in promoting tissue repair. Furthermore, recent technological developments such as single-cell RNA sequencing have allowed for unprecedented insights into the heterogeneity within the myeloid compartment, related to activation state, niche, and ontogenetic origin. A better understanding of the phenotypic and functional characteristics of macrophages critical to tissue repair and FBR processes will allow for rational design of biomaterials to promote biomaterial-tissue integration and regeneration. In this review, we discuss the role of temporal and ontogenetic macrophage heterogeneity on tissue repair processes and the FBR and the potential implications for biomaterial-directed regenerative medicine applications. STATEMENT OF SIGNIFICANCE: This review outlines the contributions of different macrophage phenotypes to different phases of wound healing and angiogenesis. Pathological outcomes, such as chronic inflammation, fibrosis, and the foreign body response, related to disruption of the macrophage inflammation-resolution process are also discussed. We summarize recent insights into the vast heterogeneity of myeloid cells related to their niche, especially the biomaterial microenvironment, and ontogenetic origin. Additionally, we present a discussion on novel tools that allow for resolution of cellular heterogeneity at the single-cell level and how these can be used to build a better understanding of macrophage heterogeneity in the biomaterial immune microenvironment to better inform immunomodulatory biomaterial design.

Immunotherapy via PD-L1-presenting biomaterials leads to long-term islet graft survival.

Antibody-mediated immune checkpoint blockade is a transformative immunotherapy for cancer. These same mechanisms can be repurposed for the control of destructive alloreactive immune responses in the transplantation setting. Here, we implement a synthetic biomaterial platform for the local delivery of a chimeric streptavidin/programmed cell death-1 (SA-PD-L1) protein to direct "reprogramming" of local immune responses to transplanted pancreatic islets. Controlled presentation of SA-PD-L1 on the surface of poly(ethylene glycol) microgels improves local retention of the immunomodulatory agent over 3 weeks in vivo. Furthermore, local induction of allograft acceptance is achieved in a murine model of diabetes only when receiving the SA-PD-L1-presenting biomaterial in combination with a brief rapamycin treatment. Immune characterization revealed an increase in T regulatory and anergic cells after SA-PD-L1-microgel delivery, which was distinct from naïve and biomaterial alone microenvironments. Engineering the local microenvironment via biomaterial delivery of checkpoint proteins has the potential to advance cell-based therapies, avoiding the need for systemic chronic immunosuppression.

Integrin-specific hydrogels modulate transplanted human bone marrow-derived mesenchymal stem cell survival, engraftment, and reparative activities.

Stem cell therapies are limited by poor cell survival and engraftment. A hurdle to the use of materials for cell delivery is the lack of understanding of material properties that govern transplanted stem cell functionality. Here, we show that synthetic hydrogels presenting integrin-specific peptides enhance the survival, persistence, and osteo-reparative functions of human bone marrow-derived mesenchymal stem cells (hMSCs) transplanted in murine bone defects. Integrin-specific hydrogels regulate hMSC adhesion, paracrine signaling, and osteoblastic differentiation in vitro. Hydrogels presenting GFOGER, a peptide targeting α2ß1 integrin, prolong hMSC survival and engraftment in a segmental bone defect and result in improved bone repair compared to other peptides. Integrin-specific hydrogels have diverse pleiotropic effects on hMSC reparative activities, modulating in vitro cytokine secretion and in vivo gene expression for effectors associated with inflammation, vascularization, and bone formation. These results demonstrate that integrin-specific hydrogels improve tissue healing by directing hMSC survival, engraftment, and reparative activities.

Lysostaphin and BMP-2 co-delivery reduces S. aureus infection and regenerates critical-sized segmental bone defects.

Staphylococcus aureus is the most common pathogen associated with bacterial infections in orthopedic procedures. Infections often lead to implant failure and subsequent removal, motivating the development of bifunctional materials that both promote repair and prevent failure due to infection. Lysostaphin is an anti-staphylococcal enzyme resulting in bacterial lysis and biofilm reduction. Lysostaphin use is limited by the lack of effective delivery methods to provide sustained, high doses of enzyme to infection sites. We engineered a BMP-2-loaded lysostaphin-delivering hydrogel that simultaneously prevents S. aureus infection and repairs nonhealing segmental bone defects in the murine radius. Lysostaphin-delivering hydrogels eradicated S. aureus infection and resulted in mechanically competent bone. Cytokine and immune cell profiling demonstrated that lysostaphin-delivering hydrogels restored the local inflammatory environment to that of a sterile injury. These results show that BMP-2-loaded lysostaphin-delivering hydrogel therapy effectively eliminates S. aureus infection while simultaneously regenerating functional bone resulting in defect healing.

Guardianship and Predatory Crimes among Incapacitated Persons in Kentucky.

Purpose: Guardianship is a legal process intended to aid incapacitated persons unable to fully care for themselves. Guardianship in the state of Kentucky ranges from total observation (24-hour care) to informal care (such as a group home) and also includes persons who are no longer being supervised but should be. One intent of guardianship includes preventing incapacitated persons from engaging in criminal acts. However, no research has been conducted on incapacitated persons currently in guardianship and the prevalence for committing predatory crimes such as murder, fire starting, or assault.Method: In this study, the authors examine guardianship supervision levels and predatory crimes in the state of Kentucky. Logistic regression analysis was used to determine the variables that significantly contributed to the model.Results: They find that unsupervised incapacitated persons are at higher odds of committing predatory crimes, leading to important policy recommendations for Kentucky guardians.

Hydrogel delivery of lysostaphin eliminates orthopedic implant infection by Staphylococcus aureus and supports fracture healing.

Orthopedic implant infections are a significant clinical problem, with current therapies limited to surgical debridement and systemic antibiotic regimens. Lysostaphin is a bacteriolytic enzyme with high antistaphylococcal activity. We engineered a lysostaphin-delivering injectable PEG hydrogel to treat Staphylococcus aureus infections in bone fractures. The injectable hydrogel formulation adheres to exposed tissue and fracture surfaces, ensuring efficient, local delivery of lysostaphin. Lysostaphin encapsulation within this synthetic hydrogel maintained enzyme stability and activity. Lysostaphin-delivering hydrogels exhibited enhanced antibiofilm activity compared with soluble lysostaphin. Lysostaphin-delivering hydrogels eradicated S. aureus infection and outperformed prophylactic antibiotic and soluble lysostaphin therapy in a murine model of femur fracture. Analysis of the local inflammatory response to infections treated with lysostaphin-delivering hydrogels revealed indistinguishable differences in cytokine secretion profiles compared with uninfected fractures, demonstrating clearance of bacteria and associated inflammation. Importantly, infected fractures treated with lysostaphin-delivering hydrogels fully healed by 5 wk with bone formation and mechanical properties equivalent to those of uninfected fractures, whereas fractures treated without the hydrogel carrier were equivalent to untreated infections. Finally, lysostaphin-delivering hydrogels eliminate methicillin-resistant S. aureus infections, supporting this therapy as an alternative to antibiotics. These results indicate that lysostaphin-delivering hydrogels effectively eliminate orthopedic S. aureus infections while simultaneously supporting fracture repair.

Three-dimensional collagen matrix induces a mechanosensitive invasive epithelial phenotype.

A critical step in breast cancer progression is local tissue invasion, during which cells pass from the epithelial compartment to the stromal compartment. We recently showed that malignant leader cells can promote the invasion of otherwise non-invasive epithelial follower cells, but the effects of this induced-invasion phenomenon on follower cell phenotype remain unclear. Notably, this process can expose epithelial cells to the stromal extracellular matrix (ECM), which is distinct from the ECM within the normal epithelial microenvironment. Here, we used a 3D epithelial morphogenesis model in which cells were cultured in biochemically and mechanically defined matrices to examine matrix-mediated gene expression and the associated phenotypic response. We found that 3D collagen matrix promoted expression of mesenchymal genes including MT1-MMP, which was required for collagen-stimulated invasive behavior. Epithelial invasion required matrix anchorage as well as signaling through Src, PI3K, and Rac1, and increasingly stiff collagen promoted dispersive epithelial cell invasion. These results suggest that leader cell-facilitated access to the stromal ECM may trigger an invasive phenotype in follower epithelial cells that could enable them to actively participate in local tissue invasion.

A prospective investigation of dietary patterns and internalizing and externalizing mental health problems in adolescents.

Investigating protective and risk factors that influence mental health in young people is a high priority. While previous cross-sectional studies have reported associations between diet and mental health among adolescents, few prospective studies exist. The aim of this study was to examine prospective relationships between dietary patterns and mental health among adolescents participating in the Western Australian Pregnancy Cohort (Raine) Study. Self-report questionnaires were used to assess indicators of mental health (Youth Self-Report externalizing/internalizing T-scores) and Western and Healthy dietary patterns (identified using factor analysis) at 14 (2003-2005) and 17 years (2006-2008). Multivariate linear and logistic regression were used to assess relationships between dietary patterns and mental health. Complete data were available for 746 adolescents. In females only, the Western dietary pattern z-score at 14 years was positively associated with greater externalizing behaviors at 17 years (ß = 1.91; 95% CI: 0.04, 3.78) and a greater odds of having clinically concerning externalizing behaviors at 17 years (OR = 1.90; 95% CI: 1.06, 3.41). No other statistically significant associations were observed. Overall our findings only lend partial support to a link between diet and mental health. We found it to be specific to females consuming a Western dietary pattern and to externalizing behaviors. Future research on dietary patterns and mental health needs to consider possible sex differences and distinguish between different mental health outcomes as well as between healthy and unhealthy dietary patterns.

Local extracellular matrix alignment directs cellular protrusion dynamics and migration through Rac1 and FAK.

Cell migration within 3D interstitial microenvironments is sensitive to extracellular matrix (ECM) properties, but the mechanisms that regulate migration guidance by 3D matrix features remain unclear. To examine the mechanisms underlying the cell migration response to aligned ECM, which is prevalent at the tumor-stroma interface, we utilized time-lapse microscopy to compare the behavior of MDA-MB-231 breast adenocarcinoma cells within randomly organized and well-aligned 3D collagen ECM. We developed a novel experimental system in which cellular morphodynamics during initial 3D cell spreading served as a reductionist model for the complex process of matrix-directed 3D cell migration. Using this approach, we found that ECM alignment induced spatial anisotropy of cells' matrix probing by promoting protrusion frequency, persistence, and lengthening along the alignment axis and suppressing protrusion dynamics orthogonal to alignment. Preference for on-axis behaviors was dependent upon FAK and Rac1 signaling and translated across length and time scales such that cells within aligned ECM exhibited accelerated elongation, front-rear polarization, and migration relative to cells in random ECM. Together, these findings indicate that adhesive and protrusive signaling allow cells to respond to coordinated physical cues in the ECM, promoting migration efficiency and cell migration guidance by 3D matrix structure.

Not Just "A Walking the Dog": Dog Walking and Pet Play and Their Association With Recommended Physical Activity Among Adolescents.

PURPOSE: To examine the role of pet play and dog walking in children's and adolescents' leisure time, and the relationship between these activities and physical activity. DESIGN: The study design was observational. SETTING: The study setting was metropolitan Perth and nonmetropolitan regions in Western Australia. SUBJECTS: The study included 1097 primary school (mean age, 10.1 years; SD, 1.6 years) and 657 secondary school (mean age, 14.0 years; SD, 1.3 years) students. MEASURES: Validated measures of total physical activity, dog walking, and pet play activity (prevalence and time) were calculated. ANALYSIS: Generalized linear models tested for differences between proportions, while adjusting for socioeconomic status, age, and school-level clustering. RESULTS: Approximately one third of primary school and one quarter of secondary school students reported that they walked the dog at least once in the last week. Pet play was the most common play activity for primary and secondary school girls, and the second and third most popular play activity for secondary and primary school boys, respectively. Secondary school students who walked the dog or played with pets spent an average of 1 hour per week on each activity, and they were significantly more likely (p < .005) to meet national physical activity recommendations than secondary school students not reporting these activities. CONCLUSION: Given the significant proportion of young people who frequently engage in dog walking and pet play, and the high level of pet ownership in many Western countries, promotion of these activities to support young people's health is warranted.