Validation of a murine proteome-wide phage display library for identification of autoantibody specificities.

Autoimmunity is characterized by loss of tolerance to tissue-specific as well as systemic antigens, resulting in complex autoantibody landscapes. Here, we introduce and extensively validate the performance characteristics of a murine proteome-wide library for phage display immunoprecipitation and sequencing (PhIP-seq) in profiling mouse autoantibodies. This library was validated using 7 genetically distinct mouse lines across a spectrum of autoreactivity. Mice deficient in antibody production (Rag2-/- and µMT) were used to model nonspecific peptide enrichments, while cross-reactivity was evaluated using anti-ovalbumin B cell receptor-restricted OB1 mice as a proof of principle. The PhIP-seq approach was then utilized to interrogate 3 distinct autoimmune disease models. First, serum from Lyn-/- IgD+/- mice with lupus-like disease was used to identify nuclear and apoptotic bleb reactivities. Second, serum from nonobese diabetic (NOD) mice, a polygenic model of pancreas-specific autoimmunity, was enriched in peptides derived from both insulin and predicted pancreatic proteins. Lastly, Aire-/- mouse sera were used to identify numerous autoantigens, many of which were also observed in previous studies of humans with autoimmune polyendocrinopathy syndrome type 1 carrying recessive mutations in AIRE. These experiments support the use of murine proteome-wide PhIP-seq for antigenic profiling and autoantibody discovery, which may be employed to study a range of immune perturbations in mouse models of autoimmunity profiling.

Molecular basis for potent B cell responses to antigen displayed on particles of viral size.

Multivalent viral epitopes induce rapid, robust and T cell-independent humoral immune responses, but the biochemical basis for such potency remains incompletely understood. We take advantage of a set of liposomes of viral size engineered to display affinity mutants of the model antigen (Ag) hen egg lysozyme. Particulate Ag induces potent 'all-or-none' B cell responses that are density dependent but affinity independent. Unlike soluble Ag, particulate Ag induces signal amplification downstream of the B cell receptor by selectively evading LYN-dependent inhibitory pathways and maximally activates NF-κB in a manner that mimics T cell help. Such signaling induces MYC expression and enables even low doses of particulate Ag to trigger robust B cell proliferation in vivo in the absence of adjuvant. We uncover a molecular basis for highly sensitive B cell responses to viral Ag display that is independent of encapsulated nucleic acids and is not merely accounted for by avidity and B cell receptor cross-linking.

Validation of a murine proteome-wide phage display library for the identification of autoantibody specificities.

Autoimmunity is characterized by loss of tolerance to tissue-specific as well as systemic antigens, resulting in complex autoantibody landscapes. Here, we introduce and extensively validate the performance characteristics of a murine proteome-wide library for phage display immunoprecipitation and sequencing (PhIP-seq), to profile mouse autoantibodies. This system and library were validated using seven genetic mouse models across a spectrum of autoreactivity. Mice deficient in antibody production (Rag2-/- and µMT) were used to model non-specific peptide enrichments, while cross-reactivity was evaluated using anti-ovalbumin B cell receptor (BCR)-restricted OB1 mice as a proof of principle. The PhIP-seq approach was then utilized to interrogate three distinct autoimmune disease models. First, serum from Lyn-/- IgD+/- mice with lupus-like disease was used to identify nuclear and apoptotic bleb reactivities, lending support to the hypothesis that apoptosis is a shared origin of these antigens. Second, serum from non-obese diabetic (NOD) mice, a polygenic model of pancreas-specific autoimmunity, enriched peptides derived from both insulin and predicted pancreatic proteins. Lastly, Aire-/- mouse sera were used to identify numerous auto-antigens, many of which were also observed in previous studies of humans with autoimmune polyendocrinopathy syndrome type 1 (APS1) carrying recessive mutations in AIRE. Among these were peptides derived from Perilipin-1, a validated autoimmune biomarker of generalized acquired lipodystrophy in humans. Autoreactivity to Perilipin-1 correlated with lymphocyte infiltration in adipose tissue and underscores the approach in revealing previously unknown specificities. These experiments support the use of murine proteome-wide PhIP-seq for antigenic profiling and autoantibody discovery, which may be employed to study a range of immune perturbations in mouse models of autoimmunity.

Molecular basis for potent B cell responses to antigen displayed on particles of viral size.

Although it has long been appreciated that multivalent antigens - and particularly viral epitope display - produce extremely rapid, robust, and T-independent humoral immune responses, the biochemical basis for such potency has been incompletely understood. Here we take advantage of a set of neutral liposomes of viral size that are engineered to display affinity mutants of the model antigen (Ag) hen egg lysozyme at precisely varied density. We show that particulate Ag display by liposomes induces highly potent B cell responses that are dose-and density-dependent but affinity-independent. Titrating dose of particulate, but not soluble, Ag reveals bimodal Erk phosphorylation and cytosolic calcium increases. Particulate Ag induces signal amplification downstream of the B cell receptor (BCR) by selectively evading LYN-dependent inhibitory pathways, but in vitro potency is independent of CD19. Importantly, Ag display on viral-sized particles signals independently of MYD88 and IRAK1/4, but activates NF- κ B robustly in a manner that mimics T cell help. Together, such biased signaling by particulate Ag promotes MYC expression and reduces the threshold required for B cell proliferation relative to soluble Ag. These findings uncover a molecular basis for highly sensitive B cell response to viral Ag display and remarkable potency of virus-like particle vaccines that is not merely accounted for by avidity and BCR cross-linking, and is independent of the contribution of B cell nucleic acid-sensing machinery.

A guide to qualitative attribution methods for evaluation in conservation.

Knowledge of what conservation interventions improve biodiversity outcomes, and in which circumstances, is imperative. Experimental and quasi-experimental methods are increasingly used to establish causal inference and build the evidence base on the effectiveness of interventions, but their ability to provide insight into how and under what conditions an intervention should be implemented to improve biodiversity outcomes faces limitations. A suite of attribution methods that leverage qualitative methods for causal inference is available but underutilized in conversation impact evaluation. This article provides a guide to 5 such qualitative attribution methods: contribution analysis, process tracing, realist evaluation, qualitative comparative analysis, and most significant change. It defines and introduces each method and then illustrates how they could be applied through a case study of community conservancies in Namibia. This guide provides examples of how qualitative attribution methods can advance knowledge of what works, in which contexts, and why in biodiversity conservation.

Guía para los métodos de atribución cualitativa para la evaluación en conservación Resumen El conocimiento sobre cuáles intervenciones de conservación son las que aumentan los resultados en biodiversidad y en cuáles circunstancias sucede esto es imperativo. Los métodos experimentales y semiexperimentales se usan cada vez más para establecer la inferencia casual y construir la base de evidencias sobre la efectividad de las intervenciones, pero su capacidad para proporcionar conocimiento sobre cómo y bajo cuáles condiciones se debería implementar una intervención para aumentar los resultados en biodiversidad enfrenta limitaciones. Un conjunto de métodos de atribución que impulse los métodos cualitativos para la inferencia casual se encuentra disponible pero poco utilizado en la evaluación de los impactos de conservación. Este artículo proporciona una guía para cinco de los métodos de atribución cualitativa: análisis de contribución, rastreo de procesos, evaluación realista, análisis cualitativo comparativo y el cambio más significativo. La guía define e introduce cada método y después ilustra cómo podrían aplicarse mediante un estudio de caso de conservación en Namibia. Esta guía proporciona ejemplos de cómo los métodos de atribución cualitativa pueden incrementar el conocimiento de qué funciona, en cuáles contextos y por qué en la conservación d la biodiversidad.

Deepwater Horizon Oil Spill Exposures and Long-term Self-rated Health Effects Among Parents in Coastal Louisiana.

PURPOSE: To assess whether exposure to the 2010 Deepwater Horizon oil spill (DHOS) was related to parents' self-rated health over time. DESIGN: 3 waves of panel data were drawn from the Gulf Coast Population Impact study (2014) and Resilient Children, Youth, and Communities study (2016, 2018). SETTING: Coastal Louisiana communities in high-impact DHOS areas. PARTICIPANTS: Respondents were parents or guardians aged 18 - 84, culled from a probability sample of households with a child aged 4 to 18 (N = 526) at the time of the 2010 DHOS. MEASURES: Self-rated health was measured at each wave. Self-reported physical exposure to the DHOS, economic exposure to the DHOS, and control variables were measured in 2014. ANALYSIS: We used econometric random effects regression for panel data to assess relationships between DHOS exposures and self-rated health over time, controlling for potentially confounding covariates. RESULTS: Both physical exposure (b = -0.39; P < 0.001) and economic exposure (b = -0.34; P < 0.001) to the DHOS had negative associations with self-rated health over the study period. Physical exposure had a larger effect size. CONCLUSION: Parents' physical contact with, and economic disruption from, the 2010 DHOS were tied to long-term diminished health.

Parental Education and Child Physical Health Following the BP Deepwater Horizon Oil Spill.

PURPOSE: To assess whether trajectories of children's physical health problems differ by parental college degree attainment in Louisiana areas highly impacted by the 2010 BP Deepwater Horizon oil spill (BP-DHOS). DESIGN: Three waves of panel data (2014, 2016, and 2018) from the Gulf Coast Population Impact / Resilient Children, Youth, and Communities studies. SETTING: BP-DHOS-impacted communities in coastal Louisiana. PARTICIPANTS: Parents of children aged 4-18 in a longitudinal probability sample (n = 392). MEASURES: Reported child physical health problems from the BP-DHOS, parental college degree attainment, and covariates. ANALYSIS: Linear growth curve models are used to assess initial levels of and the rate of change in child physical unknown. The current study uses 3 waves physical health problems by parental college degree attainment. Explanatory variables are measured at baseline and the outcome variable is measured at all 3 waves. RESULTS: Compared to children of parents without college degrees, children of college graduates had fewer initial health problems in 2014 (b = -.33; p = .02). Yet, this health advantage decreased over time, as indicated by their positive rate of change (b = .22; p = .01), such that the higher education health advantage was not statistically significant by 2018. CONCLUSION: Children of college graduates experienced a physical health advantage following the BP-DHOS, but this gap closed over time. The closure of the gap was due to the children of college graduates experiencing significant increases in reported health problems over the study period.

NR4A nuclear receptors in T and B lymphocytes: Gatekeepers of immune tolerance.

Random VDJ recombination early in T and B cell development enables the adaptive immune system to recognize a vast array of evolving pathogens via antigen receptors. However, the potential of such randomly generated TCRs and BCRs to recognize and respond to self-antigens requires layers of tolerance mechanisms to mitigate the risk of life-threatening autoimmunity. Since they were originally cloned more than three decades ago, the NR4A family of nuclear hormone receptors have been implicated in many critical aspects of immune tolerance, including negative selection of thymocytes, peripheral T cell tolerance, regulatory T cells (Treg), and most recently in peripheral B cell tolerance. In this review, we discuss important insights from many laboratories as well as our own group into the function and mechanisms by which this small class of primary response genes promotes self-tolerance and immune homeostasis to balance the need for host defense against the inherent risks posed by the adaptive immune system.

Interferon gamma constrains type 2 lymphocyte niche boundaries during mixed inflammation.

Allergic immunity is orchestrated by group 2 innate lymphoid cells (ILC2s) and type 2 helper T (Th2) cells prominently arrayed at epithelial- and microbial-rich barriers. However, ILC2s and Th2 cells are also present in fibroblast-rich niches within the adventitial layer of larger vessels and similar boundary structures in sterile deep tissues, and it remains unclear whether they undergo dynamic repositioning during immune perturbations. Here, we used thick-section quantitative imaging to show that allergic inflammation drives invasion of lung and liver non-adventitial parenchyma by ILC2s and Th2 cells. However, during concurrent type 1 and type 2 mixed inflammation, IFNγ from broadly distributed type 1 lymphocytes directly blocked both ILC2 parenchymal trafficking and subsequent cell survival. ILC2 and Th2 cell confinement to adventitia limited mortality by the type 1 pathogen Listeria monocytogenes. Our results suggest that the topography of tissue lymphocyte subsets is tightly regulated to promote appropriately timed and balanced immunity.

Health Equity in Medicine.

Description Healthcare disparities exist when, due to racial, ethnic, or gender identity differences linked to social, economic or environmental factors, certain populations lack equitable access to quality healthcare and insurance coverage. Such disparities across history carry profound future implications that we have only begun to contemplate as a profession. This special issue of the HCA Healthcare Journal of Medicine examines the critical issue of health equity in medicine and how the medical community can advance health equity through inclusive behavior and interactions in clinical and educational settings, and our communities.

Negative feedback by NUR77/Nr4a1 restrains B cell clonal dominance during early T-dependent immune responses.

B cell clones compete for entry into and dominance within germinal centers (GCs), where the highest-affinity B cell receptors (BCRs) are selected. However, diverse and low-affinity B cells can enter and reside in GCs for extended periods. To reconcile these observations, we hypothesize that a negative feedback loop may operate within B cells to preferentially restrain high-affinity clones from monopolizing the early GC niche. Here, we report a role for the nuclear receptor NUR77/Nr4a1 in this process. We show that NUR77 expression scales with antigen stimulation and restrains B cell expansion. Although NUR77 is dispensable for regulating GC size when GCs are elicited in a largely clonal manner, it serves to curb immunodominance under conditions where diverse clonal populations must compete for a constrained niche. We propose that this is important to preserve early clonal diversity in order to limit holes in the post-immune repertoire and to optimize GC selection.

The BAFFling persistence of memory B cells.

Although BAFF/BLyS and its receptor, BAFFR, play critical roles in naive B cell survival, the pathways involved in the persistence of memory B cells are largely unknown. In this issue of JEM, two groups, Müller-Winkler et al. (https://doi.org/10.1084/jem.20191393) and Lau et al. (https://doi.org/10.1084/jem.20191167), take complementary approaches to identify an essential role for BAFFR in the survival of memory B cells.

NR4A nuclear receptors restrain B cell responses to antigen when second signals are absent or limiting.

Antigen stimulation (signal 1) triggers B cell proliferation and primes B cells to recruit, engage and respond to T cell help (signal 2). Failure to receive signal 2 within a defined time window results in B cell apoptosis, yet the mechanisms that enforce dependence on co-stimulation are incompletely understood. Nr4a1-3 encode a small family of orphan nuclear receptors that are rapidly induced by B cell antigen receptor stimulation. Here, we show that Nr4a1 and Nr4a3 play partially redundant roles to restrain B cell responses to antigen in the absence of co-stimulation and do so, in part, by repressing the expression of BATF and, consequently, MYC. The NR4A family also restrains B cell access to T cell help by repressing expression of the T cell chemokines CCL3 and CCL4, as well as CD86 and ICAM1. Such NR4A-mediated regulation plays a role specifically under conditions of competition for limiting T cell help.

Hypertension as a Road to Treatment of Heart Failure with Preserved Ejection Fraction.

PURPOSE OF REVIEW: Hypertension heralds the diagnosis of heart failure (HF) with preserved ejection fraction (HFpEF) in 75-85% of cases and shares many of its adverse outcomes as well as its acute and chronic symptoms. This review provides important new data about the pathophysiology and mechanisms that connect hypertension and HFpEF as well as therapy used in both conditions. RECENT FINDINGS: The traditional model of HFpEF pathophysiology emphasizes the role of hypertension causing increased afterload on the left ventricle (LV), leading to LV hypertrophy (LVH) and subsequent LV diastolic dysfunction. Recent work has provided valuable insights into the mechanisms underlying the transition from hypertension to HFpEF, showing that the pathophysiology extends beyond LVH and diastolic dysfunction. An evolving paradigm suggests that HFpEF is inflammatory in nature with multifactorial pathophysiology, affected by age-related changes and comorbidities. Hypertension shares many of the proinflammatory mechanisms of HFpEF. Furthermore, hypertension precedes HFpEF in the majority of cases. Because of its clinically heterogeneous nature, development of standardized therapies for HFpEF has been challenging. As there are standardized approaches to hypertension, we suggest that similar approaches be used for the treatment of HFpEF, including medical and non-medical therapies. With medical therapies, a treat-to-target blood pressure (BP) strategy could be employed, such as systolic BP < 130 mmHg. With non-medical therapies, approaches to deal with physical inactivity, obesity, and sleep apnea could be used. Due to its heterogeneity, delineation of standardized therapies for HFpEF has been challenging. Focusing on the tremendous overlap of hypertensive heart disease with HFpEF, it is proposed that approaches currently used to guide therapies for hypertension be applied to the treatment of HFpEF.

Peripheral Tolerance Checkpoints Imposed by Ubiquitous Antigen Expression Limit Antigen-Specific B Cell Responses under Strongly Immunogenic Conditions.

A series of layered peripheral checkpoints maintain self-reactive B cells in an unresponsive state. Autoantibody production occurs when these checkpoints are breached; however, when and how this occurs is largely unknown. In particular, how self-reactive B cells are restrained during bystander inflammation in otherwise healthy individuals is poorly understood. A weakness has been the unavailability of methods capable of dissecting physiologically relevant B cell responses without the use of an engineered BCR. Resolving this will provide insights that decipher how this process goes awry during autoimmunity or could be exploited for therapy. In this study, we use a strong adjuvant to provide bystander innate and adaptive signals that promote B cell responsiveness in conjunction with newly developed B cell detection tools to study in detail the ways that peripheral tolerance mechanisms limit the expansion and function of self-reactive B cells activated under these conditions. We show that although self-reactive B cells are recruited into the germinal center, their development does not proceed, possibly because of rapid counterselection. Consequently, differentiation of plasma cells is blunted, and Ab responses are transient and devoid of affinity maturation. We propose this approach, and these tools can be more widely applied to track Ag-specific B cell responses to more disease-relevant Ags, without the need for BCR transgenic mice, in settings where tolerance pathways are compromised or have been genetically manipulated to drive stronger insights into the biology underlying B cell-mediated autoimmunity.

Transfer of antigen-encoding bone marrow under immune-preserving conditions deletes mature antigen-specific B cells in recipients and inhibits antigen-specific antibody production.

BACKGROUND AIMS: Pathological activation and collaboration of T and B cells underlies pathogenic autoantibody responses. Existing treatments for autoimmune disease cause non-specific immunosuppression, and induction of antigen-specific tolerance remains an elusive goal. Many immunotherapies aim to manipulate the T-cell component of T-B interplay, but few directly target B cells. One possible means to specifically target B cells is the transfer of gene-engineered BM that, once engrafted, gives rise to widespread specific and tolerogenic antigen expression within the hematopoietic system. METHODS: Gene-engineered bone marrow encoding ubiquitous ovalbumin expression was transferred after low-dose (300-cGy) immune-preserving irradiation. B-cell responsiveness was monitored by analyzing ovalbumin-specific antibody production after immunization with ovalbumin/complete Freund's adjuvant. Ovalbumin-specific B cells and their response to immunization were analyzed using multi-tetramer staining. When antigen-encoding bone marrow was transferred under immune-preserving conditions, cognate antigen-specific B cells were purged from the recipient's preexisting B-cell repertoire and the repertoire that arose after bone marrow transfer. RESULTS: OVA-specific B-cell deletion was apparent within the established host B-cell repertoire as well as that developing after gene-engineered bone marrow transfer. OVA-specific antibody production was substantially inhibited by transfer of OVA-encoding BM and activation of OVA-specific B cells, germinal center formation and subsequent OVA-specific plasmablast differentiation were all inhibited. Low levels of gene-engineered bone marrow chimerism were sufficient to limit antigen-specific antibody production. RESULTS: These data show that antigen-specific B cells within an established B-cell repertoire are susceptible to de novo tolerance induction, and this can be achieved by transfer of gene-engineered bone marrow. This adds further dimensions to the utility of antigen-encoding bone marrow transfer as an immunotherapeutic tool.

Synthetic Liposomal Mimics of Biological Viruses for the Study of Immune Responses to Infection and Vaccination.

Human viruses possess very complex supramolecular structures. Both icosahedral and enveloped viruses typically display an array of viral-encoded protein antigens at varied spatial densities on the viral particle surface. The viral nucleic acid genome, on the other hand, is encapsulated inside the viral particle. Although both the surface antigen and the interior nucleic acids could independently produce immunological responses, how B cells integrate these two types of signals and respond to a typical virus particle to initiate activation is not well understood at a molecular level. The study of these fundamental biological processes would benefit from the development of viral structural mimics that are well constructed to incorporate both quantitative and qualitative viral features for presentation to B cells. These novel tools would enable researchers to systematically dissect the underlying processes. Here we report the development of such particulate antigens based on liposomes engineered to display a model protein antigen, hen egg lysozyme (HEL). We developed methods to overexpress and purify various affinity mutants of HEL from E. coli. We conjugated the purified recombinant HEL proteins onto the surface of a virion-sized liposome in an orientation-specific manner at defined spatial densities and also encapsulated nucleic acid molecules into the interior of the liposome. Both the chemical conjugation of the HEL antigen on liposome surfaces and the encapsulation of nucleic acids were stable under physiologically relevant conditions. These liposomes elicited antigen-specific B-cell responses in vitro, which validate these supramolecular structures as a novel and effective approach to mimic and systematically isolate the role of essential viral features in directing the B-cell response to particulate antigens.

A Philanthropic Approach to Supporting Emergent Disaster Response and Recovery.

In August 2017, Hurricane Harvey struck the US Gulf Coast and caused more than US $125 billion in damages in Texas. The loss of lives and the economic damages resulted in an outpouring of support for the recovery efforts in the form of federal assistance and private donations. The latter has supported more creative approaches to recovery. Organizations that normally would not receive funding were able to obtain resources to use in novel manners. Using the framework of Dynes typology to identify groups and their respective structures and tasks, this report from the field analyzes Hurricane Harvey and the financial support mechanisms used to support recovery efforts in Texas, what organizations were funded to do, and where they fit into Dynes typology. The authors close by noting the importance of these emerging organizations and the need to support diversity in funding disaster response and recovery efforts beyond large nonprofit organizations.

Is Left Ventricular Hypertrophy a Valid Therapeutic Target?

PURPOSE OF REVIEW: The purpose of this review is to answer the question whether left ventricular hypertrophy (LVH) could be considered a therapeutic target in patients with hypertension. To fulfill this purpose, we briefly outline different methods of measuring LVH, then discuss the current evidence and unresolved controversies regarding the relationships among LVH, blood pressure (BP), and cardiovascular disease (CVD) outcomes. RECENT FINDINGS: The methods and criteria used for defining LVH in clinical studies lack consistency and are inherently different. Electrocardiogram (ECG) has been the most common method, but some studies used echocardiography, and recently, the cardiac magnetic resonance imaging was used by some studies as well. Regardless of the method, studies have shown that higher BP is a risk factor for LVH, regression of LVH is possible by successful BP lowering, and LVH is associated with CVD outcomes. Nevertheless, recent trials revealed that although intensive BP lowering (systolic BP target of < 120 mm of Hg) resulted in lower rates of developing new ECG-LVH and higher rates of regression of existing LVH, the benefit of intensive BP lowering on the risk of CV events was not meaningfully influenced by its favorable effect on ECG-LVH. These findings raise several critical questions about the mechanistic links between hypertension treatment, LVH regression, and reduction in CV events. Given these questions and findings, LVH improvement cannot yet be considered a reliable surrogate outcome measure for use in the context of hypertensive heart disease. LVH is a modifiable risk factor related to systolic BP and regression of LVH may reduce subsequent CV events. However, LVH may not be the "holy grail" in regard to therapeutic targets in hypertensive heart disease, but it could be considered one of the markers in the successful management of hypertension.

Re-examining balinese subaks through the lens of cultural multilevel selection.

Overcoming environmental challenges requires understanding when and why individuals adopt cooperative behaviors, how individual behaviors and interactions among resource users change over time, and how group structure and group dynamics impact behaviors, institutions, and resource conditions. Cultural multilevel selection (CMLS) is a theoretical framework derived from theories of cultural evolution and cultural group selection that emphasizes pressures affecting different levels of social organization as well as conflicts among these levels. As such, CMLS can be useful for understanding many environmental challenges. With this paper, we use evidence from the literature and hypothetical scenarios to show how the framework can be used to understand the emergence and persistence of sustainable social-ecological systems. We apply the framework to the Balinese system of rice production and focus on two important cultural traits (synchronized cropping and the institutions and rituals associated with water management). We use data from the literature that discusses bottom-up (self-organized, complex adaptive system) and top-down explanations for the system and discuss how (1) the emergence of group structure, (2) group-level variation in cropping strategies, institutions, and rituals, and (3) variation in overall yields as a result of different strategies and institutions, could have allowed for the spread of group-beneficial traits and the increasing complexity of the system. We also outline cultural transmission mechanisms that can explain the spread of group-beneficial traits in Bali and describe the kinds of data that would be required to validate the framework in forward-looking studies.