Integrating population and single-cell variations in vaccine responses identifies a naturally adjuvanted human immune setpoint.

Multimodal single-cell profiling methods can capture immune cell variations unfolding over time at the molecular, cellular, and population levels. Transforming these data into biological insights remains challenging. Here, we introduce a framework to integrate variations at the human population and single-cell levels in vaccination responses. Comparing responses following AS03-adjuvanted versus unadjuvanted influenza vaccines with CITE-seq revealed AS03-specific early (day 1) response phenotypes, including a B cell signature of elevated germinal center competition. A correlated network of cell-type-specific transcriptional states defined the baseline immune status associated with high antibody responders to the unadjuvanted vaccine. Certain innate subsets in the network appeared "naturally adjuvanted," with transcriptional states resembling those induced uniquely by AS03-adjuvanted vaccination. Consistently, CD14+ monocytes from high responders at baseline had elevated phospho-signaling responses to lipopolysaccharide stimulation. Our findings link baseline immune setpoints to early vaccine responses, with positive implications for adjuvant development and immune response engineering.

Hypoxic treatment of human dual placental perfusion induces a preeclampsia-like inflammatory response.

Preeclampsia is a human pregnancy-specific disorder characterized by a placental pro-inflammatory response in combination with an imbalance of angiogenic factors and clinical symptoms, including hypertension and proteinuria. Insufficient uteroplacental oxygenation in preeclampsia due to impaired trophoblast invasion during placentation is believed to be responsible for many of the molecular events leading to the clinical manifestations of this disease. We investigated the use of hypoxic treatment of the dual placental perfusion system as a model for preeclampsia. A modified perfusion technique allowed us to achieve a mean soluble oxygen tension within the intervillous space (IVS) of 5-7% for normoxia and <3% for hypoxia (as a model for preeclampsia). We assayed for the levels of different inflammatory cytokines, oxidative stress markers, as well as other factors, such as endothelin (ET)-1 that are known to be implicated as part of the inflammatory response in preeclampsia. Our results show a significant increase under hypoxia in the levels of different inflammatory cytokines, including IL-6 (P=0.002), IL-8 (P<0.0001), TNF-α (P=0.032) and IFN-γ (P=0.009) at 360 min in maternal venous samples (n=6). There was also a significant increase in ET-1 levels under hypoxia both on the maternal side at 30 min (P=0.003) and fetal side at 360 min (P=0.036) (n=6). Other markers of oxidative stress, including malondialdehyde and 8-iso-protaglandin F2α (P=0.009) also show increased levels. Overall, these findings indicate that exposure of ex vivo dually perfused placental tissue to hypoxia provides a useful model for mimicking the inflammatory response characteristic of preeclampsia. This would therefore provide a powerful tool for studying and further delineating the molecular mechanisms involved in the underlying pathophysiology of preeclampsia.

Telehealth Expansion, Internet Speed, and Primary Care Access Before and During COVID-19.

Importance: Primary care (PC) receipt is associated with better health outcomes. How telehealth expansion and internet speed are associated with PC use is unclear. Objective: To examine the association of telehealth and internet speed with PC use across sociodemographic determinants of health. Design, Setting, and Participants: This cohort study performed difference-in-differences regression of the change in in-person and telehealth PC visits between pre-COVID-19 public health emergency (PHE) (June 1, 2019, to February 29, 2020) and an initial (March 1, 2020, to May 31, 2020) and prolonged (March 1, 2020, to December 31, 2021) PHE period among continuously enrolled nonpregnant, nondisabled Wisconsin Medicaid beneficiaries aged 18 to 64 years. Data were analyzed from March 2022 to March 2023. Exposure: PHE-induced telehealth expansion. Main Outcomes and Measures: Change in PC telehealth (using Current Procedural Terminology codes) visits: (1) count; (2) visit share completed by telehealth; (3) percentage of PHE-induced visit decline offset by telehealth. High-speed internet (HSI) defined as living in a census block group with a median block maximum download speed of 940 megabits per second or greater (June 2020 Federal Communications Commission broadband data); other census block groups classified as low-speed internet (LSI). Results: In the total cohort of 172 387 participants, 102 989 (59.7%) were female, 103 848 (60.2%) were non-Hispanic White, 34 258 (19.9%) were non-Hispanic Black, 15 020 (8.7%) were Hispanic, 104 239 (60.5%) were aged 26 to 45 years, and 112 355 (66.0%) lived in urban counties. A total of 142 433 (82.6%) had access to HSI; 72 524 (42.1%) had a chronic condition. There was a mean (SD) of 0.138 (0.261) pre-PHE PC visits per month. In the pre-PHE period, visit rates were significantly higher for female than male participants, non-Hispanic White than non-Hispanic Black individuals, urban than rural residents, those with HSI than LSI, and patients with chronic disease than patients without. In the initial PHE period, female participants had a greater increase in telehealth visits than male participants (43.1%; 95% CI, 37.02%-49.18%; P < .001), share (2.20 percentage point difference [PPD]; 95% CI, 1.06-3.33 PPD; P < .001) and offset (6.81 PPD; 95% CI, 3.74-9.87 PPD; P < .001). Non-Hispanic Black participants had a greater increase in share than non-Hispanic White participants (5.44 PPD; 95% CI, 4.07-6.81 PPD; P < .001) and offset (15.22 PPD; 95% CI, 10.69-19.75 PPD; P < .001). Hispanic participants had a greater increase in telehealth visits than Non-Hispanic White participants (35.60%; 95% CI, 25.55%-45.64%; P < .001), share (8.50 PPD; 95% CI, 6.75-10.26 PPD; P < .001) and offset (12.93 PPD; 95% CI, 6.25-19.60 PPD; P < .001). Urban participants had a greater increase in telehealth visits than rural participants (63.87%; 95% CI, 52.62%-75.11%; P < .001), share (9.13 PPD; 95% CI, 7.84-10.42 PPD; P < .001), and offset (13.31 PPD; 95% CI; 9.62-16.99 PPD; P < .001). Participants with HSI had a greater increase in telehealth visits than those with LSI (55.23%; 95% CI, 42.26%-68.20%; P < .001), share (6.61 PPD; 95% CI, 5.00-8.23 PPD; P < .001), and offset (6.82 PPD; 95% CI, 2.15-11.49 PPD; P = .004). Participants with chronic disease had a greater increase in telehealth visits than those with none (188.07%; 95% CI, 175.27%-200.86%; P < .001), share (4.50 PPD; 95% CI, 3.58-5.42 PPD; P < .001), and offset (9.03 PPD; 95% CI, 6.01-12.04 PPD; P < .001). Prolonged PHE differences were similar. Differences persisted among those with HSI. Conclusions and Relevance: In this cohort study of Wisconsin Medicaid beneficiaries, greater telehealth uptake occurred in groups with higher pre-PHE utilization, except for high uptake among Hispanic and non-Hispanic Black individuals despite low pre-PHE utilization. HSI did not moderate disparities. These findings suggest telehealth and HSI may boost PC receipt, but will generally not close utilization gaps.

Endothelin-1-induced endoplasmic reticulum stress in disease.

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) represents a cellular stress induced by multiple stimuli and pathologic conditions. Recent evidence implicates endothelin-1 (ET-1) in the induction of placental ER stress in pregnancy disorders. ER stress has previously also been implicated in various other disease states, including neurodegenerative disorders, diabetes, and cardiovascular diseases, as has ET-1 in the pathophysiology of these conditions. However, to date, there has been no investigation of the link between ET-1 and the induction of ER stress in these disease states. Based on recent evidence and mechanistic insight into the role of ET-1 in the induction of placental ER stress, the following review attempts to outline the broader implications of ET-1-induced ER stress, as well as strategies for therapeutic intervention based around ET-1.

Endothelin-1 induces endoplasmic reticulum stress by activating the PLC-IP(3) pathway: implications for placental pathophysiology in preeclampsia.

Recent evidence implicates placental endoplasmic reticulum (ER) stress in the pathophysiological characteristics of preeclampsia. Herein, we investigate whether endothelin (ET)-1, which induces Ca(2+) release from the ER, can induce placental ER stress. Loss of ER Ca(2+) homeostasis impairs post-translational modification of proteins, triggering ER stress-response pathways. IHC confirmed the presence of both ET-1 and its receptors in the syncytiotrophoblast. Protein levels and immunoreactivity of ET-1 and the endothelin B receptor (ETBR) were increased in preeclamptic samples compared with normotensive controls. JEG-3 and BeWo choriocarcinoma cells treated with ET-1 displayed an increase in ER stress markers. ET-1 induced phospho-activation of the ETBR. Treating cells with BQ788, an ETBR antagonist, or small-interfering RNA knockdown of the receptor inhibited induction of ER stress. ET-1 also stimulated p-phospholipase C (PLC)γ1 levels. By using inhibitors of PLC activation, U73122, and the inositol 1,4,5-triphosphate (IP(3)) receptor, xestospongin-C, we demonstrated that ET-1 induces ER stress via the PLC-IP(3) pathway. Furthermore, ET-1 levels increased in the syncytiotrophoblast of explants from normal placentas after hypoxia-reoxygenation in vitro. Conditioned medium from hypoxia-reoxygenation explants also contained higher ET-1 levels, which induced ER stress in JEG-3 cells that was abolished by an ET-1-neutralizing antibody. Collectively, the data show that ET-1 induced ER stress in trophoblasts via the ETBR and initiation of signaling through the PLC-IP(3) pathway, with the potential for autocrine stimulation.

Increased placental phospholipid levels in pre-eclamptic pregnancies.

Physiological pregnancy is associated with an increase in lipids from the first to the third trimester. This is a highly regulated response to satisfy energy and membrane demands of the developing fetus. Pregnancy disorders, such as pre-eclampsia, are associated with a dysregulation of lipid metabolism manifesting in increased maternal plasma lipid levels. In fetal placental tissue, only scarce information on the lipid profile is available, and data for gestational diseases are lacking. In the present study, we investigated the placental lipid content in control versus pre-eclamptic samples, with the focus on tissue phospholipid levels and composition. We found an increase in total phospholipid content as well as changes in individual phospholipid classes in pre-eclamptic placental tissues compared to controls. These alterations could be a source of placental pathological changes in pre-eclampsia, such as lipid peroxide insult or dysregulation of lipid transport across the syncytiotrophoblast.

ET-traps: Potential therapeutics for preeclampsia.

Elevated endothelin-1 (ET-1) has been implicated in several diseases including preeclampsia, where it causes the induction of hypertension, oxidative stress, endoplasmic reticulum stress, microvascular dysfunction and tissue damage in different organs. ET-traps are Fc-fusion proteins with a design based on the physiological receptors of ET-1. This paper discusses the potential use of ET-traps as a therapeutic for preeclampsia. ET-traps potently bind and sequester pathologically elevated ET-1 to significantly reduce different markers of pathology to non-disease levels with no toxicity.

Multiscale integration of human and single-cell variations reveals unadjuvanted vaccine high responders are naturally adjuvanted.

Advances in multimodal single cell analysis can empower high-resolution dissection of human vaccination responses. The resulting data capture multiple layers of biological variations, including molecular and cellular states, vaccine formulations, inter- and intra-subject differences, and responses unfolding over time. Transforming such data into biological insight remains a major challenge. Here we present a systematic framework applied to multimodal single cell data obtained before and after influenza vaccination without adjuvants or pandemic H5N1 vaccination with the AS03 adjuvant. Our approach pinpoints responses shared across or unique to specific cell types and identifies adjuvant specific signatures, including pro-survival transcriptional states in B lymphocytes that emerged one day after vaccination. We also reveal that high antibody responders to the unadjuvanted vaccine have a distinct baseline involving a rewired network of cell type specific transcriptional states. Remarkably, the status of certain innate immune cells in this network in high responders of the unadjuvanted vaccine appear "naturally adjuvanted": they resemble phenotypes induced early in the same cells only by vaccination with AS03. Furthermore, these cell subsets have elevated frequency in the blood at baseline and increased cell-intrinsic phospho-signaling responses after LPS stimulation ex vivo in high compared to low responders. Our findings identify how variation in the status of multiple immune cell types at baseline may drive robust differences in innate and adaptive responses to vaccination and thus open new avenues for vaccine development and immune response engineering in humans.

Endothelin-1: a key pathological factor in pre-eclampsia?

Endothelin (ET)-1 has been implicated in a diverse range of signalling events in a wide variety of target tissues. Given its potent vasoactive function and the prevalence of hypertension in pre-eclampsia, there has been extensive research on the role of ET-1 in this disorder. Indeed, ET-1 has been suggested to contribute to hypertension in pre-eclampsia. Recently, ET-1 has also been implicated in the induction of both oxidative stress and endoplasmic reticulum stress in pre-eclampsia; each of which has been proposed to contribute to many of the clinical manifestations of this disorder. ET-1 has been shown to activate key signalling molecules that lead to induction of these stress pathways. The use of ET-receptor antagonists could block oxidative and endoplasmic reticulum stress. Hence, further research into the role of ET-1 in pre-eclampsia may lead to the development of possible strategies to circumvent these stress pathways and the associated pathology that occurs in pre-eclampsia. Endothelin (ET)-1 has been implicated in a diverse range of signalling events in a wide variety of target tissues. Given its potent vasoactive function and the prevalence of hypertension in pre-eclampsia, there has been extensive research on the role of ET-1 in this disorder. Indeed, ET-1 has been suggested to contribute to hypertension in pre-eclampsia. Recently, ET-1 has also been implicated in the induction of both oxidative stress and endoplasmic reticulum stress in pre-eclampsia, each of which has been proposed to contribute to many of the clinical manifestations of this disorder. ET-1 has been shown to activate key signalling molecules that lead to induction of these stress pathways. The use of ET-receptor antagonists could block oxidative and endoplasmic reticulum stress. Hence, further research into the role of ET-1 in pre-eclampsia may lead to the development of possible strategies to circumvent these stress pathways and the associated pathology that occurs in pre-eclampsia.

Investigating the specificity of endothelin-traps as a potential therapeutic tool for endothelin-1 related disorders.

BACKGROUND: Endothelin (ET)-traps are Fc-fusion proteins with a design based on the physiological receptors of ET-1. Previous work has shown that use of the selected ET-traps potently and significantly reduces different markers of diabetes pathology back to normal, non-disease levels. AIM: To demonstrate the selected ET-traps potently and significantly bind to ET-1. METHODS: We performed phage display experiments to test different constructs of ET-traps, and conducted bio-layer interferometry binding assays to verify that the selected ET-traps bind specifically to ET-1 and display binding affinity in the double-digit picomolar range (an average of 73.8 rM, n = 6). RESULTS: These experiments have confirmed our choice of the final ET-traps and provided proof-of-concept for the potential use of constructs as effective biologics for diseases associated with pathologically elevated ET-1. CONCLUSION: There is increased need for such therapeutics as they could help save millions of lives around the world.

ET-traps offer a potential therapeutic tool for use in different autoimmune diseases.

Research shows that endothelin (ET)-traps are a potential therapy for diabetes. Given that type 1 diabetes mellitus (T1DM) is an autoimmune disorder, ET-traps could also have an efficacious, therapeutic effect on other autoimmune diseases associated with pathologically elevated ET-1. Here, we describe those different autoimmune diseases that might benefit from a tool such as ET-traps, which potently sequester these elevated levels of ET-1. We also discuss the current use of ET receptor (ETR) antagonists and the associated adverse effects, and how ET-traps are associated with no toxicity and potentially offer a superior alternative. ET-traps could be used against different autoimmune diseases and, therefore, are a novel therapeutic tool for such conditions.

Endothelin-1 traps as a potential therapeutic tool: from diabetes to beyond?

There is substantial research on the vasoactive peptide endothelin (ET)-1 in physiology, as well as in pathology. In fact, pathologically elevated levels of ET-1 have been found in several disease states, such as various cardiovascular diseases, different cancers, some neurodegenerative disorders, as well as in diabetes. Here, we describe and discuss ET-1, its importance in different diseases, and the potential therapeutic effects of ET-traps in the treatment of these diseases. Previous in vitro and in vivo research (in the diabetes disease space) demonstrated that ET-traps potently and significantly prevent the induction of different markers of diabetes-related pathology. This included induction of extracellular matrix (ECM) proteins (collagen 4α1 and fibronectin), which are pathologically elevated in diabetes. The ET-traps prevented induction of these and brought a significant return to non-diabetic levels. We also discuss the merits of using ET-traps over the currently used endothelin receptor antagonists (ERAs) and previously used therapeutic antibodies.

In vivo studies demonstrate that endothelin-1 traps are a potential therapy for type I diabetes.

BACKGROUND: Type 1 diabetes is a serious, lifelong condition where the body's blood glucose level increases because of the body's inability to make insulin. An important consequence of this is the increased expression of extracellular matrix proteins, such as fibronectin and collagen 4α1, in key tissues and organs like the heart and kidneys. Diabetes is also associated with increased plasma levels of the vasoactive peptide endothelin (ET)-1. This further aggravates the expression of the ECM proteins. There are also important consequences of increased glucose and ET-1 levels in diabetes on the heart, termed diabetic cardiomyopathy. METHODS: We have previously reported the development of ET-traps, which potently and significantly reduce pathological levels of ET-1. In this study, we tested the in vivo therapeutic potential of ET-traps for type 1 diabetes using the B6 mouse model. RESULTS: Following subcutaneous administration of ET-traps 3 times a week, over a 2 month period, the 500 nM dose of ET-traps gave a significant reduction in collagen 4α1 expression in the heart and kidney, returning it back to control, non-diabetic levels at both the mRNA and protein levels. The expression of fibronectin mRNA is also returned to control levels with the 500 nM dose of ET-traps. The efficacy of ET-traps for type 1 diabetes was further evinced by immunohistochemistry data, echocardiography studies (measuring left ventricular systolic function and diastolic dysfunction) and a measure of urine creatinine and albumin levels. In all analyses, the 500 nM dose of ET-traps returns the different measures to control, non-diabetic levels. CONCLUSION: Data from this study show that in a mouse model ET-traps have a potent and significant therapeutic effect on diabetes disease pathology. Future studies could further evaluate the use of ET-traps as a therapy for diabetes, including taking them through clinical trials.

Endothelin-1 traps potently reduce pathologic markers back to basal levels in an in vitro model of diabetes.

BACKGROUND: Diabetes mellitus is a group of metabolic disorders in which there are high blood glucose levels over a prolonged period. Diabetes is one of many diseases associated with pathologically elevated levels of endothelin (ET)-1. We have recently proposed the development of ET-traps, which are an antibody - based fusion protein that potently bind and sequester pathologically elevated levels of endothelin-1. METHODS: We constructed ET-traps that were found to be very potent binders to ET-1, with a KD of 32.5ρM. We then treated human retinal microvascular endothelial cells (HRMECs), which are an in vitro model of glucose induced cellular damage, with 10 nM ET-1 or high glucose levels (25 mM). RESULTS: In this study, we investigated the effects of our ET-trap constructs on the expression levels of both collagen 4α1 and fibronectin, which are both important pathologic markers in diabetes. Treating HRMECs with 10 nM ET-1 or 25 mM glucose significantly induces the expression of the ECM proteins fibronectin and collagen 4α1, as is found in chronic diabetic complications; Incubation of the cells with the ET-traps significantly prevented the increased expression of fibronectin and collagen 4α1 back to basal levels. This was found with both mRNA and protein expression levels of the two ECM proteins. CONCLUSION: Our results provide the first evidence of the efficacy of ET-traps in reducing pathologic markers in an in vitro model (of diabetes). Further research is warranted to determine the efficacy of ET-traps as a therapeutic tool for diabetes, which is a major public health burden around the world.