Childhood contact with social services and risk of suicide or sudden death in young adulthood: identifying hidden risk in a population-wide cohort study.

BACKGROUND: Childhood out-of-home care is associated with premature death in adulthood, in particular death by suicide, accidents and violence. However, little is known about the mortality risk in the much larger population of adults that had contact with social services in childhood but never entered out-of-home care. We determine the association between all tiers of contact with children's social services and risk of suicide and other sudden deaths in young adulthood. METHODS: This population-wide, longitudinal, record-linkage study of adults in Northern Ireland born between 1985 and 1997 (n=437 008) followed each individual from age 18 years to July 2021 (maximum age 36 years). Cox regression models estimated the association between level of contact with social services in childhood (no contact; referred but assessed as not in need (NIN); child in need (CIN) and child in care (CIC)) and risk of death by suicide and sudden death in young adulthood. RESULTS: Individuals with childhood social care contact (n=51 097) comprised 11.7% of the cohort yet accounted for 35.3% of sudden deaths and 39.7% of suicide deaths. Risk of suicide or sudden death increased stepwise with level of childhood contact and was highest in adults with a history of out-of-home care (suicide HR 8.85 (95% CI 6.83 to 11.4)). Individuals assessed as NIN, and those deemed a CIN, had four times the risk of death by suicide in young adulthood compared with unexposed peers (HR 4.25 (95% CI 3.26 to 5.53) and HR 4.49 (95% CI 3.75 to 5.39), respectively). CONCLUSION: Childhood contact with social services is a risk marker for death by suicide and sudden death in young adulthood. Risk is not confined to adults with a history of out-of-home care but extends to the much larger population that had contact with social services but never entered care.

The mental health of all children in contact with social services: a population-wide record-linkage study in Northern Ireland.

AIMS: Children in contact with social services are at high risk for mental ill health, but it is not known what proportion of the child population has contact with social services or how risk varies within this group compared to unexposed peers. We aim to quantify the extent and nature of contact with social services within the child population in Northern Ireland (NI) and the association with mental ill health. We also examine which social care experiences identify those most at risk. METHODS: This is a population-based record-linkage study of 497,269 children (aged under 18 years) alive and resident in NI in 2015 using routinely collected health and social care data. Exposure was categorized as (1) no contact, (2) referred but assessed as not in need (NIN), (3) child in need (CIN) and (4) child in care (CIC). Multilevel logistic regression analyses estimated odds ratios (ORs) for mental ill health indicated by receipt of psychotropic medication (antidepressants, anxiolytics, antipsychotics and hypnotics), psychiatric hospital admission and hospital-presenting self-harm or ideation. RESULTS: Over one in six children (17.2%, n = 85,792) were currently or previously in contact with social services, and almost one child in every 20 (4.8%, n = 23,975) had contact in 2015. Likelihood of any mental ill health outcome increased incrementally with the level of contact with social services relative to unexposed peers: NIN (OR 5.90 [95% confidence interval (CI) 5.10-6.83]), CIN (OR 5.99 [95% CI 5.50-6.53]) and CIC (OR 12.60 [95% CI 10.63-14.95]). All tiers of contact, number of referrals, number of care episodes and placement type were strongly associated with the likelihood of mental ill health. CONCLUSION: Children who have contact with social services account for a large and disproportionate amount of mental ill health in the child population. Likelihood of poor mental health across indicators is highest in care experienced children but also extends to the much larger population of children in contact with social services but never in care. Findings suggest a need for targeted mental health screening and enhanced support for all children in contact with social services.

Homologous recombination deficiency in newly diagnosed FIGO stage III/IV high-grade epithelial ovarian cancer: a multi-national observational study.

OBJECTIVE: Olaparib plus bevacizumab maintenance therapy improves survival outcomes in women with newly diagnosed, advanced, high-grade ovarian cancer with a deficiency in homologous recombination. We report data from the first year of routine homologous recombination deficiency testing in the National Health Service (NHS) in England, Wales, and Northern Ireland between April 2021 and April 2022. METHODS: The Myriad myChoice companion diagnostic was used to test DNA extracted from formalin-fixed, paraffin-embedded tumor tissue in women with newly diagnosed International Federation of Gynecology and Obstetrics (FIGO) stage III/IV high-grade epithelial ovarian, fallopian tube, or primary peritoneal cancer. Tumors with homologous recombination deficiency were those with a BRCA1/2 mutation and/or a Genomic Instability Score (GIS) ≥42. Testing was coordinated by the NHS Genomic Laboratory Hub network. RESULTS: The myChoice assay was performed on 2829 tumors. Of these, 2474 (87%) and 2178 (77%) successfully underwent BRCA1/2 and GIS testing, respectively. All complete and partial assay failures occurred due to low tumor cellularity and/or low tumor DNA yield. 385 tumors (16%) contained a BRCA1/2 mutation and 814 (37%) had a GIS ≥42. Tumors with a GIS ≥42 were more likely to be BRCA1/2 wild-type (n=510) than BRCA1/2 mutant (n=304). The distribution of GIS was bimodal, with BRCA1/2 mutant tumors having a higher mean score than BRCA1/2 wild-type tumors (61 vs 33, respectively, χ2 test p<0.0001). CONCLUSION: This is the largest real-world evaluation of homologous recombination deficiency testing in newly diagnosed FIGO stage III/IV high-grade epithelial ovarian, fallopian tube, or primary peritoneal cancer. It is important to select tumor tissue with adequate tumor content and quality to reduce the risk of assay failure. The rapid uptake of testing across England, Wales, and Northern Ireland demonstrates the power of centralized NHS funding, center specialization, and the NHS Genomic Laboratory Hub network.

Predicting Clinical Remission of Chronic Urticaria Using Random Survival Forests: Machine Learning Applied to Real-World Data.

INTRODUCTION: The time required to reach clinical remission varies in patients with chronic urticaria (CU). The objective of this study is to develop a predictive model using a machine learning methodology to predict time to clinical remission for patients with CU. METHODS: Adults with ≥ 2 ICD-9/10 relevant CU diagnosis codes/CU-related treatment > 6 weeks apart were identified in the Optum deidentified electronic health record dataset (January 2007 to June 2019). Clinical remission was defined as ≥ 12 months without CU diagnosis/CU-related treatment. A random survival forest was used to predict time from diagnosis to clinical remission for each patient based on clinical and demographic features available at diagnosis. Model performance was assessed using concordance, which indicates the degree of agreement between observed and predicted time to remission. To characterize clinically relevant groups, features were summarized among cohorts that were defined based on quartiles of predicted time to remission. RESULTS: Among 112,443 patients, 73.5% reached clinical remission, with a median of 336 days from diagnosis. From 1876 initial features, 176 were retained in the final model, which predicted a median of 318 days to remission. The model showed good performance with a concordance of 0.62. Patients with predicted longer time to remission tended to be older with delayed CU diagnosis, and have more comorbidities, more laboratory tests, higher body mass index, and polypharmacy during the 12-month period before the first CU diagnosis. CONCLUSIONS: Applying machine learning to real-world data enabled accurate prediction of time to clinical remission and identified multiple relevant demographic and clinical variables with predictive value. Ongoing work aims to further validate and integrate these findings into clinical applications for CU management.

Racing against time: leveraging preclinical models to understand pulmonary susceptibility to perinatal acetaminophen exposures.

Over the past decade, clinicians have increasingly prescribed acetaminophen (APAP) for patients in the neonatal intensive care unit (NICU). Acetaminophen has been shown to reduce postoperative opiate burden, and may provide similar efficacy for closure of the patent ductus arteriosus (PDA) as nonsteroidal anti-inflammatory drugs (NSAIDs). Despite these potential benefits, APAP exposures have spread to increasingly less mature infants, a highly vulnerable population for whom robust pharmacokinetic and pharmacodynamic data for APAP are lacking. Concerningly, preclinical studies suggest that perinatal APAP exposures may result in unanticipated adverse effects that are unique to the developing lung. In this review, we discuss the clinical observations linking APAP exposures to adverse respiratory outcomes and the preclinical data demonstrating a developmental susceptibility to APAP-induced lung injury. We show how clinical observations linking perinatal APAP exposures to pulmonary injury have been taken to the bench to produce important insights into the potential mechanisms underlying these findings. We argue that the available data support a more cautious approach to APAP use in the NICU until large randomized controlled trials provide appropriate safety and efficacy data.

Lower threshold to NFκB activity sensitizes murine ß-cells to streptozotocin.

The ß-cell response to injury may be as critical for the development of diabetes as the specific insult. In the current study, we used streptozotocin (STZ) to injure the ß-cell in order to study the response with a focus on NFκB. MIN6 cells were exposed to STZ (0.5-8 mM, 0-24h) ±TNFα (100 ng/mL) and ±IκBß siRNA to lower the threshold to NFκB activation. Cell viability was determined by trypan blue exclusion. NFκB activation was determined by the expression of the target genes Nos2 and Cxcl10, localization of the NFκB proteins p65 and p50, and expression and localization of the NFκB inhibitors, IκBß and IκBα. There was no NFκB activation in MIN6 cell exposed to STZ (2 mM) alone. However, knocking down IκBß expression using siRNA resulted in STZ-induced expression of NFκB target genes and increased cell death, while co-incubation with STZ and TNFα enhanced cell death compared to either exposure alone. Adult male IκBß-/- and WT mice were exposed to STZ and monitored for diabetes. The IκBß-/- mice developed hyperglycemia and diabetes more frequently than controls following STZ exposure. Based on these results we conclude that STZ exposure alone does not induce NFκB activity. However, lowering the threshold to NFκB activation by co-incubation with TNFα or lowering IκBß levels by siRNA sensitizes the NFκB response to STZ and results in a higher likelihood of developing diabetes in vivo. Therefore, increasing the threshold to NFκB activation through stabilizing NFκB inhibitory proteins may prevent ß-cell injury and the development of diabetes.

Real-World Burden of Nonalcoholic Steatohepatitis.

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) is associated with an increase in healthcare resource use and poor health-related quality of life (HRQoL). We assessed the humanistic and economic burden of NASH, disease management, and patient journey. METHODS: We performed a cross-sectional analysis of data, collected from July through November 2017, from the Growth from Knowledge Disease Atlas Real-World Evidence program, reported by physicians in United States, France, and Germany. We extracted demographic and medical data from medical records. Some patients voluntarily completed a survey that provided information on disease history, treatment satisfaction, and patient-reported outcomes. RESULTS: We analyzed data from 1216 patients (mean age, 54.9±12.3 years; 57.5% male; mean body mass index, 31.7±6.9); 64.6% had biopsy-confirmed NASH and comorbidities were recorded for 41.3%. Treatments included lifestyle modification (64.6%) or use of statins (25.0%), vitamin E (23.5%), or metformin (20.2%). Patients with biopsy-confirmed NASH reported more physician (4.5 vs 3.7) and outpatient visits (1.8 vs1.4) than patients with suspected NASH not confirmed by biopsy. Among the 299 patients who completed the survey, 47.8% reported various symptoms associated to their NASH. Symptomatic patients reported significantly lower HRQoL than patients without symptoms. CONCLUSIONS: In an analysis of data from 3 countries, we found NASH to be associated with regular use of medical resources; patients with symptoms of NASH had reduced HRQoL. The burden of NASH appears to be underestimated. Studies are needed to determine the burden of NASH by fibrosis stage and disease severity.

CpG-ODN-mediated TLR9 innate immune signalling and calcium dyshomeostasis converge on the NFκB inhibitory protein IκBß to drive IL1α and IL1ß expression.

Sterile inflammation contributes to many pathological states associated with mitochondrial injury. Mitochondrial injury disrupts calcium homeostasis and results in the release of CpG-rich mitochondrial DNA. The role of CpG-stimulated TLR9 innate immune signalling and sterile inflammation is well studied; however, how calcium dyshomeostasis affects this signalling is unknown. Therefore, we interrogated the relationship beτween intracellular calcium and CpG-induced TLR9 signalling in murine macrophages. We found that CpG-ODN-induced NFκB-dependent IL1α and IL1ß expression was significantly attenuated by both calcium chelation and calcineurin inhibition, a finding mediated by inhibition of degradation of the NFκB inhibitory protein IκBß. In contrast, calcium ionophore exposure increased CpG-induced IκBß degradation and IL1α and IL1ß expression. These results demonstrate that through its effect on IκBß degradation, increased intracellular Ca2+ drives a pro-inflammatory TLR9-mediated innate immune response. These results have implications for the study of innate immune signalling downstream of mitochondrial stress and injury.

Clinical Interventions to Prevent Tumour Lysis Syndrome in Hematologic Malignancy: A Multisite Retrospective Chart Review.

BACKGROUND: Tumour lysis syndrome (TLS) occurs when lysis of malignant cells causes electrolyte disturbances and potentially organ dysfunction. Guidelines recommending preventive therapy according to TLS risk are based on low-quality evidence. OBJECTIVES: The primary objective was to characterize utilization of TLS preventive strategies through comprehensive description of current practice. Secondary objectives were to determine TLS incidence, to compare use of preventive strategies among intermediate- and high-risk patients, and to describe TLS treatment strategies. METHODS: This retrospective chart review examined data for patients with newly diagnosed hematologic malignancy who were admitted to an oncology centre and/or affiliated intensive care unit between October 2015 and September 2016 in Toronto, Ontario, Canada. RESULTS: Fifty-eight patients (29 at intermediate risk, 29 at high risk) were eligible for inclusion. Use of preventive allopurinol, IV bicarbonate, and furosemide was similar between groups. Rasburicase was more frequently used for high-risk patients (3% [1/29] of intermediate-risk patients versus 36% [9/25] of high-risk patients; p = 0.003). In 4 (14%) of the intermediate-risk patients and 2 (8%) of the high-risk patients, TLS developed during the admission. TLS was observed in 10% (1/10) of patients who received preventive rasburicase and 11% (5/44) of those who did not (p > 0.99), and in 9% (4/45) of patients who received preventive IV bicarbonate and 25% (2/8) of those who did not (p = 0.22). Treatment strategies included rasburicase, IV bicarbonate, furosemide, and renal replacement therapy. CONCLUSIONS: In this retrospective chart review, rasburicase was more commonly used for high-risk patients, whereas the use of other agents was similar between risk groups. This pattern of use is inconsistent with guidelines, which recommend that all high-risk patients receive rasburicase. There was no difference in TLS incidence between patients who did and did not receive preventive rasburicase or IV bicarbonate. Further prospective studies are needed to inform management of patients with malignancies who are at intermediate or high risk of TLS.

CONTEXTE: Le syndrome de lyse tumorale (SLT) se produit lorsque la lyse de cellules malignes provoque des perturbations électrolytiques et la dysfonction potentielle d'un organe. Les lignes directrices préconisant une thérapie préventive basée sur le risque de SLT se fondent sur des éléments de preuve de piètre qualité. OBJECTIFS: L'objectif principal consistait à décrire l'adoption des stratégies de prévention du SLT en décrivant précisément la pratique actuelle. Les objectifs secondaires consistaient, quant à eux, à déterminer l'incidence du SLT, à comparer l'utilisation des stratégies de prévention pour les patients présentant un risque élevé et moyen et à décrire les stratégies de traitement du SLT. MÉTHODES: Cet examen rétrospectif a permis d'examiner les données de patients ayant récemment reçu un diagnostic d'hémopathie maligne et ayant été admis dans un centre d'oncologie ou une unité de soins intensifs affiliée, entre octobre 2015 et septembre 2016 à Toronto (Ontario), au Canada. RÉSULTATS: Cinquante-huit patients (29 présentant un risque moyen et 29 un risque élevé) étaient admissibles. L'utilisation d'allopurinol à titre préventif, de bicarbonate par voie intraveineuse et de furosémide était similaire d'un groupe à l'autre. Le rasburicase était plus fréquemment utilisé pour les patients présentant un risque élevé (3 % [1/29] de patients présentant un risque moyen contre 36 % [9/25] de patients présentant un risque élevé; p = 0.003). Quatre (14 %) patients présentant un risque moyen et deux (8 %) présentant un risque élevé ont développé un SLT pendant l'admission. Le SLT a été observé chez 10 % (1/10) des patients ayant reçu du rasburicase à titre préventif et chez 11 % (5/44) des patients qui n'en avaient pas reçu (p > 0,99); il a aussi été observé chez 9 % (4/45) des patients ayant reçu du bicarbonate par voie intraveineuse à titre préventif et chez 25 % (2/8) des patients qui n'en avaient pas reçu (p = 0.22). Les stratégies de traitement comprenaient le rasburicase, le bicarbonate par voie intraveineuse, le furosémide et la thérapie de remplacement rénal. CONCLUSIONS: Dans cet examen rétrospectif des dossiers, l'usage du rasburicase était plus fréquent pour les patients présentant un risque élevé, tandis que celui d'autres agents était similaire entre les groupes à risque. Ce schéma d'utilisation n'est pas conforme aux lignes directrices, qui recommandent que tous les patients présentant un risque élevé reçoivent du rasburicase. Aucune différence n'est apparue dans l'incidence du SLT parmi les patients ayant reçu du rasburicase ou du bicarbonate par voie intraveineuse à titre préventif et parmi ceux qui n'en avaient pas reçu. Davantage d'études prospectives sont nécessaires pour mieux connaitre la gestion des patients à haut risque ou ceux qui présentent des risques moyens de SLT, mais qui ont des malignités.

Developmentally Regulated Innate Immune NFκB Signaling Mediates IL-1α Expression in the Perinatal Murine Lung.

Bronchopulmonary dysplasia (BPD) is the most common morbidity complicating premature birth. Importantly, preclinical models have demonstrated that IL-1 receptor antagonism prevents the lung injury and subsequent abnormal development that typically results following perinatal exposure to inflammatory stresses. This receptor is activated by two pro-inflammatory cytokines, IL-1α and IL-1ß. While many studies have linked IL-1ß to BPD development, IL-1α is relatively under-studied. The objective of our study was to determine whether systemic inflammatory stress induces IL-1α expression in the neonatal lung, and if so, whether this expression is mediated by innate immune NFκB signaling. We found that endotoxemia induced IL-1α expression during the saccular stage of neonatal lung development and was not present in the other neonatal organs or the adult lung. This IL-1α expression was dependent upon sustained pulmonary NFκB activation, which was specific to the neonatal lung. Using in vivo and in vitro approaches, we found that pharmacologic and genetic inhibition of NFκB signaling attenuated IL-1α expression. These findings demonstrate that innate immune regulation of IL-1α expression is developmentally regulated and occurs via an NFκB dependent mechanism. Importantly, the specific role of developmentally regulated pulmonary IL-1α expression remains unknown. Future studies must determine the effect of attenuating innate immune IL-1α expression in the developing lung before adopting broad IL-1 receptor antagonism as an approach to prevent neonatal lung injury.

Sex-differences in LPS-induced neonatal lung injury.

Being of the male sex has been identified as a risk factor for multiple morbidities associated with preterm birth, including bronchopulmonary dysplasia (BPD). Exposure to inflammatory stress is a well-recognized risk factor for developing BPD. Whether there is a sex difference in pulmonary innate immune TLR4 signaling, lung injury and subsequent abnormal lung development is unknown. Neonatal (P0) male and female mice (ICR) were exposed to systemic LPS (5 mg/kg, IP) and innate immune signaling, and the transcriptional response were assessed (1 and 5 hours), along with lung development (P7). Male and female mice demonstrated a similar degree of impaired lung development with decreased radial alveolar counts, increased surface area, increased airspace area and increased mean linear intercept. We found no differences between male and female mice in the baseline pulmonary expression of key components of TLR4-NFκB signaling, or in the LPS-induced pulmonary expression of key mediators of neonatal lung injury. Finally, we found no difference in the kinetics of LPS-induced pulmonary NFκB activation between male and female mice. Together, these data support the conclusion that the innate immune response to early postnatal LPS exposure and resulting pulmonary sequelae is similar in male and female mice.

The hepatic innate immune response is lobe-specific in a murine model endotoxemia.

The liver plays a central role in the innate immune response to endotoxemia. While previous studies have demonstrated lobe-specific transcriptional responses to various insults, whether this is true in response to endotoxemia is unknown. We sought to assess whether there were significant intra- and inter-lobe differences in the murine hepatic innate immune transcriptional response to endotoxemia. Adult male ICR mice were exposed to i.p. LPS (5 mg/kg, 30 min, 60 min, 5 h) and primary ( Tnf, Cxcl1, Nfkbia, Tnfiap3) and secondary ( Il6, Nos2) innate immune response gene expression was assessed in the left medial, right medial, left lateral, and right lateral lobes, and the papillary and caudate processes. The expression of all innate immune response genes increased following i.p. LPS challenge. When tested at the early time points (30 and 60 min), the left medial lobe and caudate process consistently demonstrated the highest induction of gene expression. Most inter-lobe differences were attenuated at later time points (5 h). To improve reproducibility of the study of endotoxemia induced by i.p. LPS challenge, inclusion of appropriate methodological details regarding collection of hepatic tissue should be included when reporting scientific results in published manuscripts.

Immunotolerant p50/NFκB Signaling and Attenuated Hepatic IFNß Expression Increases Neonatal Sensitivity to Endotoxemia.

Sepsis is a major cause of neonatal morbidity and mortality. The current paradigm suggests that neonatal susceptibility to infection is explained by an innate immune response that is functionally immature. Recent studies in adults have questioned a therapeutic role for IFNß in sepsis; however, the role of IFNß in mediating neonatal sensitivity to sepsis is unknown. We evaluated the transcriptional regulation and expression of IFNß in early neonatal (P0) and adult murine models of endotoxemia (IP LPS, 5 mg/kg). We found that hepatic, pulmonary, and serum IFNß expression was significantly attenuated in endotoxemic neonates when compared to similarly exposed adults. Furthermore, endotoxemia induced hepatic p65/NFκB and IRF3 activation exclusively in adults. In contrast, endotoxemia induced immunotolerant p50/NFκB signaling in neonatal mice without evidence of IRF3 activation. Consistent with impaired IFNß expression and attenuated circulating serum levels, neonatal pulmonary STAT1 signaling and target gene expression was significantly lower than adult levels. Using multiple in vivo approaches, the source of hepatic IFNß expression in endotoxemic adult mice was determined to be the hepatic macrophage, and experiments in RAW 264.7 cells confirmed that LPS-induced IFNß expression was NFκB dependent. Finally, treating neonatal mice with IFNß 2 h after endotoxemia stimulated pulmonary STAT1 signaling and STAT1 dependent gene expression. Furthermore, IFNß treatment of endotoxemic neonatal animals resulted in significantly improved survival following exposure to lethal endotoxemia. In conclusion, endotoxemia induced IFNß expression is attenuated in the early neonatal period, secondary to impaired NFκB-p65/IRF3 signaling. Pre-treatment with IFNß decreases neonatal sensitivity to endotoxemia. These results support further study of the role of impaired IFNß expression and neonatal sensitivity to sepsis.

Macrophage-derived IL-1ß/NF-κB signaling mediates parenteral nutrition-associated cholestasis.

In infants intolerant of enteral feeding because of intestinal disease, parenteral nutrition may be associated with cholestasis, which can progress to end-stage liver disease. Here we show the function of hepatic macrophages and phytosterols in parenteral nutrition-associated cholestasis (PNAC) pathogenesis using a mouse model that recapitulates the human pathophysiology and combines intestinal injury with parenteral nutrition. We combine genetic, molecular, and pharmacological approaches to identify an essential function of hepatic macrophages and IL-1ß in PNAC. Pharmacological antagonism of  IL-1 signaling or genetic deficiency in CCR2, caspase-1 and caspase-11, or IL-1 receptor (which binds both IL-1α and IL-1ß) prevents PNAC in mice. IL-1ß increases hepatocyte NF-κB signaling, which interferes with farnesoid X receptor and liver X receptor bonding to respective promoters of canalicular bile and sterol transporter genes (Abcc2, Abcb11, and Abcg5/8), resulting in transcriptional suppression and subsequent cholestasis. Thus, hepatic macrophages, IL-1ß, or NF-κB may be targets for restoring bile and sterol transport to treat PNAC.

Redistribution of Extracellular Superoxide Dismutase Causes Neonatal Pulmonary Vascular Remodeling and PH but Protects Against Experimental Bronchopulmonary Dysplasia.

BACKGROUND: A naturally occurring single nucleotide polymorphism (SNP), (R213G), in extracellular superoxide dismutase (SOD3), decreases SOD3 matrix binding affinity. Humans and mature mice expressing the R213G SNP exhibit increased cardiovascular disease but decreased lung disease. The impact of this SNP on the neonatal lung at baseline or with injury is unknown. METHODS: Wild type and homozygous R213G mice were injected with intraperitoneal bleomycin or phosphate buffered saline (PBS) three times weekly for three weeks and tissue harvested at 22 days of life. Vascular and alveolar development were evaluated by morphometric analysis and immunostaining of lung sections. Pulmonary hypertension (PH) was assessed by right ventricular hypertrophy (RVH). Lung protein expression for superoxide dismutase (SOD) isoforms, catalase, vascular endothelial growth factor receptor 2 (VEGFR2), endothelial nitric oxide synthase (eNOS) and guanosine triphosphate cyclohydrolase-1 (GTPCH-1) was evaluated by western blot. SOD activity and SOD3 expression were measured in serum. RESULTS: In R213G mice, SOD3 lung protein expression decreased, serum SOD3 protein expression and SOD serum activity increased compared to wild type (WT) mice. Under control conditions, R213G mice developed pulmonary vascular remodeling (decreased vessel density and increased medial wall thickness) and PH; alveolar development was similar between strains. After bleomycin injury, in contrast to WT, R213G mice were protected from impaired alveolar development and their vascular abnormalities and PH did not worsen. Bleomycin decreased VEGFR2 and GTPCH-1 only in WT mice. CONCLUSION: R213G neonatal mice demonstrate impaired vascular development and PH at baseline without alveolar simplification, yet are protected from bleomycin induced lung injury and worsening of pulmonary vascular remodeling and PH. These results show that vessel bound SOD3 is essential in normal pulmonary vascular development, and increased serum SOD3 expression and SOD activity prevent lung injury in experimental bronchopulmonary dysplasia (BPD) and PH.

Inhibition of IκBß/NFκB signaling prevents LPS-induced IL1ß expression without increasing apoptosis in the developing mouse lung.

BackgroundThe pro-inflammatory consequences of IL1ß expression contribute to the pathogenesis of bronchopulmonary dysplasia. Selectively targeting Lipopolysaccharide (LPS)-induced IκBß/NFκB signaling attenuates IL1ß mRNA expression in macrophages. Whether targeting IκBß/NFκB signaling affects the anti-apoptotic gene expression, a known consequence of global LPS-induced NFκB inhibition, is unknown.MethodsMacrophages (RAW 264.7, bone marrow-derived macrophage) were assessed for LPS-induced IL1ß mRNA/protein expression, anti-apoptotic gene expression, cell viability (trypan blue exclusion), and activation of apoptosis (caspase-3 and PARP cleavage) following pharmacologic and genetic attenuation of IκBß/NFκB signaling. Expressions of IL1ß and anti-apoptotic genes were assessed in endotoxemic newborn mice (P0) with intact (WT), absent (IκBß KO), and attenuated (IκBß overexpressing) IκBß/NFκB signaling.ResultsIn cultured macrophages, pharmacologic and genetic inhibition of LPS-induced IκBß/NFκB signaling significantly attenuated IL1ß mRNA and protein expression. Importantly, targeting IκBß/NFκB signaling did not attenuate LPS-induced expression of anti-apoptotic genes or result in cell death. In endotoxemic neonatal mice, targeting LPS-induced IκBß/NFκB signaling significantly attenuated pulmonary IL1ß expression without affecting the anti-apoptotic gene expression.ConclusionTargeting IκBß/NFκB signaling prevents LPS-induced IL1ß expression without inducing apoptosis in cultured macrophages and in the lungs of endotoxemic newborn mice. Inhibiting this pathway may prevent inflammatory injury without affecting the protective role of NFκB activity in the developing lung.

Perinatal Endotoxemia Induces Sustained Hepatic COX-2 Expression through an NFκB-Dependent Mechanism.

BACKGROUND: Exposure to perinatal infection is associated with the multiple morbidities complicating preterm birth. How a relatively immature innate immune response contributes to this is unknown. OBJECTIVE: We sought to determine if the perinatal innate immune response to endotoxemia induces a unique pattern of cyclooxygenase-2 (COX-2) expression via an NFκB-dependent mechanism. METHODS: Hepatic and pulmonary COX-2 mRNA expression was assessed following perinatal (at embryonic days 15 and 19 and after birth) or adult endotoxemia. Hepatic NFκB activity was assessed by cytosolic inhibitory protein degradation and subunit nuclear translocation. Immunohistochemistry and isolated cell preparations determined hepatic macrophage COX-2 expression, and the effect of pharmacologic and genetic inhibition of NFκB activity was tested. RESULTS: Perinatal endotoxemia induced sustained hepatic macrophage COX-2 expression and NFκB activity compared to in exposed adults. Isolated hepatic macrophages and immunohistochemistry demonstrated enriched LPS-induced COX-2 expression that was sensitive to pharmacologic and genetic approaches to attenuate NFκB activity. Finally, pharmacologic inhibition of endotoxemia-induced NFκB activity in neonatal mice prevented hepatic NFκB activity and attenuated COX-2 expression. CONCLUSION: Our findings of sustained neonatal hepatic NFκB activity and COX-2 expression in response to endotoxemia support a robust perinatal innate immune response. This may represent a link between the innate immune response and the pathogenesis of diseases associated with preterm birth.

Endotoxemia Induces IκBß/NF-κB-Dependent Endothelin-1 Expression in Hepatic Macrophages.

Elevated serum concentrations of the vasoactive protein endothelin-1 (ET-1) occur in the setting of systemic inflammatory response syndrome and contribute to distal organ hypoperfusion and pulmonary hypertension. Thus, understanding the cellular source and transcriptional regulation of systemic inflammatory stress-induced ET-1 expression may reveal therapeutic targets. Using a murine model of LPS-induced septic shock, we demonstrate that the hepatic macrophage is the primary source of elevated circulating ET-1, rather than the endothelium as previously proposed. Using pharmacologic inhibitors, ET-1 promoter luciferase assays, and by silencing and overexpressing NF-κB inhibitory protein IκB expression, we demonstrate that LPS-induced ET-1 expression occurs via an NF-κB-dependent pathway. Finally, the specific role of the cRel/p65 inhibitory protein IκBß was evaluated. Although cytoplasmic IκBß inhibits activity of cRel-containing NF-κB dimers, nuclear IκBß stabilizes NF-κB/DNA binding and enhances gene expression. Using targeted pharmacologic therapies to specifically prevent IκBß/NF-κB signaling, as well as mice genetically modified to overexpress IκBß, we show that nuclear IκBß is both necessary and sufficient to drive LPS-induced ET-1 expression. Together, these results mechanistically link the innate immune response mediated by IκBß/NF-κB to ET-1 expression and potentially reveal therapeutic targets for patients with Gram-negative septic shock.

Inhibiting IκBß-NFκB signaling attenuates the expression of select pro-inflammatory genes.

Multiple mediators of septic shock are regulated by the transcription factor nuclear factor κB (NFκB). However, complete NFκB inhibition can exacerbate disease, necessitating evaluation of targeted strategies to attenuate the pro-inflammatory response. Here, we demonstrate that in murine macrophages, low-dose NFκB inhibitors specifically attenuates lipopolysaccharide (LPS)-induced IκBß degradation and the expression of a select subset of target genes (encoding IL1ß, IL6, IL12ß). Gain- and loss-of-function experiments demonstrate the necessary and sufficient role of inhibitor of NFκB family member IκBß (also known as NFKBIB) in the expression of these genes. Furthermore, both fibroblasts and macrophages isolated from IκBß overexpressing mice demonstrate attenuated LPS-induced IκBß-NFκB signaling and IL1ß, IL6 and IL12ß expression. Further confirming the role of IκBß and its NFκB subunit binding partner cRel in LPS-induced gene expression, pre-treatment of wild-type mouse embryonic fibroblasts with a cell-permeable peptide containing the cRel nuclear localization sequence attenuated IL6 expression. We prove that LPS-induced IκBß-NFκB signaling can be selectively modulated to attenuate the expression of select pro-inflammatory target genes, thus providing therapeutic insights for patients exposed to systemic inflammatory stress.

Wnt5a is necessary for normal kidney development in zebrafish and mice.

BACKGROUND: Wnt5a is important for the development of various organs and postnatal cellular function. Little is known, however, about the role of Wnt5a in kidney development, although WNT5A mutations were identified in patients with Robinow syndrome, a genetic disease which includes developmental defects in kidneys. Our goal in this study was to determine the role of Wnt5a in kidney development. METHODS: Whole-mount in situ hybridization was used to establish the expression pattern of Wnt5a during kidney development. Zebrafish with wnt5a knockdown and Wnt5a global knockout mice were used to identify kidney phenotypes. RESULTS: In zebrafish, wnt5a knockdown resulted in glomerular cyst formation and dilated renal tubules. In mice, Wnt5a global knockout resulted in pleiotropic, but severe, kidney phenotypes, including agenesis, fused kidney, hydronephrosis and duplex kidney/ureter. CONCLUSIONS: Our data demonstrated the important role of Wnt5a in kidney development. Disrupted Wnt5a resulted in kidney cysts in zebrafish and pleiotropic abnormal kidney development in mice.