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Background Neonatal hyperbilirubinemia is one of the most common conditions for neonate inpatients. Indonesia faces a major challenge in which different guidelines regarding the management of this condition were present. This study aimed to compare the existing guidelines regarding prevention, diagnosis, treatment and monitoring in order to create the best recommendation for a new hyperbilirubinemia guideline in Indonesia. Methods Through an earlier survey regarding adherence to the neonatal hyperbilirubinemia guideline, we identified that three main guidelines are being used in Indonesia. These were developed by the Indonesian Pediatric Society (IPS), the Ministry of Health (MoH), and World Health Organization (WHO). In this study, we compared factors such as prevention, monitoring, methods for identifying, risk factors in the development of neonatal jaundice, risk factors that increase brain damage, and intervention treatment threshold in the existing guidelines to determine the best recommendations for a new guideline. Results The MoH and WHO guidelines allow screening and treatment of hyperbilirubinemia based on visual examination (VE) only. Compared with the MoH and WHO guidelines, risk assessment is comprehensively discussed in the IPS guideline. The MoH guideline recommends further examination of an icteric baby to ensure that the mother has enough milk without measuring the bilirubin level. The MoH guideline recommends referring the baby when it looks yellow on the soles and palms. The WHO and IPS guidelines recommend combining VE with an objective measurement of transcutaneous or serum bilirubin. The threshold to begin phototherapy in the WHO guideline is lower than the IPS guideline while the exchange transfusion threshold in both guidelines are comparably equal. Conclusions The MoH guideline is outdated. MoH and IPS guidelines are causing differences in approaches to the management hyperbilirubinemia. A new, uniform guideline is required.

Highlights : • Risk factors that can increase the incidence of CLM in humans include male sex, children aged

Parkinson's disease is a chronic progressive neurodegenerative disease of the extrapyramidal system. These symptoms are divided into specific stages based on the severity of the symptoms. Differences in Parkinson's disease severity are linked to the quality of life in Parkinson's disease patients. Demonstrate the relationship between stage and attitude quality in Parkinson's disease patients, Department of Neurology, Muhammadiyah Malang University Hospital. Thirty-seven respondents were obtained from the analytical observations using a crossover design nonrandom objective sampling method. The Hoehn and Yahr Scale for Diagnosing Parkinson's Disease Stage and Parkinson's Disease Questionnaire (PDQ-39) was used to assess the quality of the attitude process in patients with Parkinson's disease during the previous month. The Spearman Rank correction test result is 0.485, which reduces the correction between street and road quality. It is unidirectional because the correlation value is positive, so the higher the ladder, the lower the patient's quality of life. The adjustment between significant variables was 0.002 (α

The function of cognition in young adults (around the age of 20 years) mostly does not develop to its peak, even though at that age a person needs better cognitive abilities to deal with the lectures. Brain training game activities by playing games through the NeuronationTM application can improve cognitive function. To determine the effect of Brain training activities on improvement of cognitive function. Experimental study with two group pre and post design. 74 Sample were medical students of Faculty of Medicine, University of Muhammadiyah Malang class of 2017-2018, divided into control and treatment groups of 37 samples each. The treatment group was given by game brain game training 30 minutes a day, 20 times in 4 weeks.. Cognitive function was measured by the MoCA-Ina test in the control group and pre and post test. The hypothesis test used the pairet samples test. Cognitive function of pre and post test of the control group good cognitive function increased 2.69% and cognitive function of pre and post test of treatment group increased 58.8% with the result of pairet sample analysis of significant p = 0,000 which means that in the control group there was a tendency for increased function cognition after treatment (post test) but the improvement was not significant. In the treatment group with brain training activities cognitive function increased sharply and statistically significant, Brain training activities affect the improvement of cognitive function.

Background Necrotizing enterocolitis (NEC) is a prematurity-related complication of the gastrointestinal tract that affects 3-15% of preterm infants. Due to its atypical signs and symptoms, NEC is often diagnosed late, leading to mortality and morbidity. Objective To describe the incidence, characteristics, and survival rate of preterm infants with NEC in the Neonatal Unit of Cipto Mangunkusumo Hospital. Methods This prospective cohort study was conducted on preterm infants born in Cipto Mangunkusumo Hospital in 2019 who had NEC Bell stage 2 or higher. Subjects were recruited consecutively. NEC was classified into either early-onset (

Background Preterm infants are susceptible to the damaging effects of hyperoxia which may lead to bronchopulmonary dysplasia (BPD) and intestinal damage. Hyperoxia also affects intestinal microbiota. The optimal initial FiO2 for the resuscitation of premature infants is unknown. Objective To determine the effect of different initial oxygen concentrations on BPD, oxidative stress markers, damage to the gastrointestinal mucosa, and the intestinal microbiome. Methods We conducted an unblinded, randomized controlled clinical trial in premature infants requiring supplemental oxygen in the first minutes of life. Infants started at an FiO2 of either 30% (low) or 50% (moderate), which was adjusted to achieve target oxygen saturations (SpO2) of 88-92% by 10 minutes of life using pulse oximetry. The primary outcome was incidence of BPD. Secondary outcomes included markers of oxidative stress [oxidized glutathione (GSH)/reduced glutathione (GSSG) ratio and malondialdehyde (MDA)], intestinal integrity indicated by fecal alpha-1 antitrypsin (AAT), and intestinal microbiota on fecal examination. Results Eighty-four infants were recruited. There was no significant difference in rates of BPD between the 30% FiO2 and 50% FiO2 groups (42.8% vs. 40.5%, respectively). Nor were there significant differences in GSH/GSSG ratios, MDA concentrations, fecal AAT levels, or changes in facultative anaerobic and anaerobic microbiota between groups. Conclusion In premature infants resuscitated using low vs. moderate initial FiO2 levels, we find no significant differences in BPD incidence, markers of oxidative stress, intestinal mucosa integrity, or intestinal microbiota.

Background: Hyperglycemia condition will decline cognitive function. No basic therapy has been found for this. Purple potato anthocyanins are useful as anti-inflammatory, antioxidant, neuroprotectant, and antidiabetic. Objective: Evaluate effect of purple potato’s anthocyanins on MDA levels, brain’s caspase-3 expressiom, and spatial memory function in diabetic model of Wistar rats. Methods: This is an experimental study using diabetic model rats. The sample was divided into negative and positive control, anthocyanin dose of 10mg/kg, 20mg/kg, and 80mg/kg groups. MDA levels were measured using spectrophotometer, caspase-3 expression with immunohistochemistry, and spatial memory function using Morris water maze test. Results: Tukey test showed that anthocyanin 10, 20, and 80 mg/kg lowering MDA levels, caspase-3 expression, and Morris water maze’s travel time compared to control positive (p = 0.000). But anthocyanin 80 mg/kg make a significant increase on these three variabels compared to 10 and 20 mg/kg groups (p = 0.010). Pearson test showed that there no correlation between anthocyanin’s dose, MDA levels, caspase-3 expression, and Morris water maze test. Conclusion: Anthocyanin doses 10 and 20mg/kg lowering MDA levels and caspase-3 expression, also improves spatial memory function on diabetic model of Wistar rats.

Background: The CDC and ACIP recommend COVID-19 vaccination for patients with inborn errors of immunity (IEI). Not much is known about vaccine safety in IEI, and whether vaccination attenuates infection severity in IEI., Objective: To estimate COVID-19 vaccination safety and examine effect on outcomes in patients with IEI., Methods: We built a secure registry database in conjunction with the US Immunodeficiency Network to examine vaccination frequency and indicators of safety and effectiveness in IEI patients. The registry opened on January 1, 2022, and closed on August 19, 2022., Results: Physicians entered data on 1245 patients from 24 countries. The most common diagnoses were antibody deficiencies (63.7%). At least one COVID-19 vaccine was administered to 806 patients (64.7%), and 216 patients received vaccination prior to the development of COVID-19. The most common vaccines administered were mRNA-based (84.0%). Seventeen patients were reported to seek outpatient clinic or emergency room care for a vaccine-related complication, and one patient was hospitalized for symptomatic anemia. Eight hundred twenty-three patients (66.1%) experienced COVID-19 infection. Of these, 156 patients required hospitalization (19.0%), 47 required ICU care (5.7%), and 28 died (3.4%). Rates of hospitalization (9.3% versus 24.4%, p < 0.001), ICU admission (2.8% versus 7.6%, p = 0.013), and death (2.3% versus 4.3%, p = 0.202) in patients who had COVID-19 were lower in patients who received vaccination prior to infection. In adjusted logistic regression analysis, not having at least one COVID-19 vaccine significantly increased the odds of hospitalization and ICU admission., Conclusion: Vaccination for COVID-19 in the IEI population appears safe and attenuates COVID-19 severity., (© 2024. The Author(s).)

Background: As the burden of allergic and immunologic disease continues to increase, there is increased demand for a larger Allergy and Immunology (AI) subspecialty workforce. The field must prioritize the expansion of our workforce and the recruitment of exceptional and diverse trainees to ensure the vitality of the specialty. Although the AI fellowship match has traditionally been competitive, recent trends in fellowship applications have demonstrated fewer applicants per fellowship position. This trend has made recruitment a priority on the agenda of the national AI societies., Objective: To elucidate key factors influencing the decision to choose the field of AI by querying fellows-in-training., Methods: A survey was created and distributed yearly to fellows-in-training from 2017 to 2021 to identify factors influencing a career choice in AI., Results: Approximately 59% of respondents rotated with AI in residency and 35% in both medical school and residency. Most respondents reported having a mentor in the field before fellowship, and many had their first exposures to AI during medical school (40%) or residency (32%). Most respondents decided to pursue AI during residency. The most common factors that influenced the decision to pursue AI were work/life balance, clinical aspects of the field, mentorship, and research opportunities., Conclusions: Our data suggest that the decision to pursue a career in AI often occurs during residency training and is motivated primarily by work/life balance, clinical aspects of the field, and clinician mentorship. Our survey results could provide guidance to AI training programs on strategies to recruit exceptional and diverse trainees., (Copyright © 2024 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: The mechanism for anaphylaxis following mRNA COVID-19 vaccination has been widely debated; understanding this serious adverse event is important for future vaccines of similar design. A mechanism proposed is type I hypersensitivity (i.e., IgE-mediated mast cell degranulation) to polyethylene glycol (PEG). Using an assay that, uniquely, had been previously assessed in patients with anaphylaxis to PEG, our objective was to compare anti-PEG IgE in serum from mRNA COVID-19 vaccine anaphylaxis case-patients and persons vaccinated without allergic reactions. Secondarily, we compared anti-PEG IgG and IgM to assess alternative mechanisms., Methods: Selected anaphylaxis case-patients reported to U.S. Vaccine Adverse Event Reporting System December 14, 2020-March 25, 2021 were invited to provide a serum sample. mRNA COVID-19 vaccine study participants with residual serum and no allergic reaction post-vaccination ("controls") were frequency matched to cases 3:1 on vaccine and dose number, sex and 10-year age category. Anti-PEG IgE was measured using a dual cytometric bead assay (DCBA). Anti-PEG IgG and IgM were measured using two different assays: DCBA and a PEGylated-polystyrene bead assay. Laboratorians were blinded to case/control status., Results: All 20 case-patients were women; 17 had anaphylaxis after dose 1, 3 after dose 2. Thirteen (65 %) were hospitalized and 7 (35 %) were intubated. Time from vaccination to serum collection was longer for case-patients vs controls (post-dose 1: median 105 vs 21 days). Among Moderna recipients, anti-PEG IgE was detected in 1 of 10 (10 %) case-patients vs 8 of 30 (27 %) controls (p = 0.40); among Pfizer-BioNTech recipients, it was detected in 0 of 10 case-patients (0 %) vs 1 of 30 (3 %) controls (p >n 0.99). Anti-PEG IgE quantitative signals followed this same pattern. Neither anti-PEG IgG nor IgM was associated with case status with both assay formats., Conclusion: Our results support that anti-PEG IgE is not a predominant mechanism for anaphylaxis post-mRNA COVID-19 vaccination., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Mary A Staat reports a relationship with Pfizer that includes: funding grants. Mary A Staat reports a relationship with Merck & Co Inc that includes: funding grants. Mary A Staat reports a relationship with UpToDate that includes: consulting or advisory. Donna S. Hummell reports a relationship with Merck & Co Inc that includes: consulting or advisory. Kathryn M. Edwards reports a relationship with Bionet that includes: consulting or advisory. Kathryn M. Edwards reports a relationship with IBM that includes: consulting or advisory. Kathryn M. Edwards reports a relationship with Member Data that includes: consulting or advisory. Kathryn M. Edwards reports a relationship with Sanofi, X-4 Pharma, Seqirus, Moderna, Pfizer, Merck, Roache, Novavax, Brighton Collaboration that includes: consulting or advisory. UpToDate fees for Mary A. Stadt were classified as royalties that are unrelated to this work. Consultative work by Kathryn M. Edwards for X-4 Pharma, Seqirus, Moderna, Pfizer, Merck, Roche, Novavax, Brighton Collaboration was serving on Safety and Monitoring Boards. Consultative work by Donna S. Hummel for Merck was for eDMC monitoring for clinical trial., (Published by Elsevier Ltd.)

Background: Amoxicillin-associated reactions (AARs) contribute to substantial health care utilization, with a reutilization rate of 10% in pediatric emergency department (ED) and urgent care (UC) settings., Objective: To identify predictors of ED/UC reutilization by examining patients' clinical features and providers' management of AARs., Methods: Through a retrospective chart review of 668 patients presenting with AARs over 2 years to the pediatric ED/UC, we examined clinical features associated with ED/UC reutilization, including rash phenotype, systemic symptoms (fever, angioedema, joint involvement, gastrointestinal symptoms), and providers' management (pharmacologic treatment and counseling). We then constructed a statistical model to predict ED/UC reutilization using stepwise backward model selection., Results: ED/UC reutilizers were more likely to be male (P = .008) and have fever (P = .0001), angioedema (P < .0001), joint involvement (P < .0001), and gastrointestinal symptoms (P = .0001) during their AAR course. Rash phenotypes differed between groups (P < .0001), as ED/UC reutilizers more frequently exhibited urticaria. However, there were no differences in clinical management between groups, including pharmacologic recommendations, at the initial ED/UC encounter. In addition, our statistical model identified younger patients <2 years of age as more likely to reutilize ED/UC resources if providers did not document specific return precautions (odds ratio, 3.6; 95% confidence interval, 1.7-7.7)., Conclusion: Recognition of clinical features and treatment gaps associated with ED/UC reutilization will guide interventions to optimize care in children presenting with AARs, such as improved anticipatory guidance and early allergy consultation. Prospective studies are needed to determine whether these interventions will reduce ED/UC reutilization and facilitate timely allergy testing., (Copyright © 2022 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Amoxicillin-associated reactions (AARs) in children presenting as rashes are common, and recent data suggest that >90% tolerate amoxicillin on re-exposure. However, additional data would help pediatricians and allergists gain confidence in referring and testing children who experienced systemic symptoms perceived as "worrisome," thus leading to urgent medical evaluations. By characterizing the entire spectrum of AAR symptoms in pediatric patients presenting to emergency department (ED)/urgent care (UC) settings, we sought to increase our diagnostic acumen to guide subsequent allergy evaluations., Objective: To fully characterize clinical features of rash and systemic symptoms in children presenting to the ED/UC with AARs., Methods: A retrospective chart review of children seen in the ED/UC from July 1, 2015, to June 30, 2017, was conducted. Clinical features, chronology, and seasonality were detailed, and cases were classified into 3 previously described AAR phenotypes: maculopapular exanthem (MPE), urticaria, and serum sickness-like reactions (SSLRs), if they experienced joint symptoms., Results: Children (n = 668; median age: 1.8 years) presented to the ED/UC with urticaria (44%), MPE (36%), and SSLRs (11%) typically on days 7 to 10 of amoxicillin. Although children with SSLRs were more frequently treated with corticosteroids (28%, P < .0001) and exhibited higher rates of "worrisome" features (fever, angioedema, or gastrointestinal symptoms; 73%, P < .0001), delayed-onset systemic symptoms were identified frequently in all 3 groups. ED/UC reutilization was unexpectedly high with 66 children (10%) returning to the ED/UC for re-evaluation., Conclusion: "Worrisome" symptoms are common in children presenting to the ED/UC with AARs. Future studies are needed to determine the impact on subsequent referral and allergy testing., (Copyright © 2022 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Recurrent and life-threatening respiratory infections are nearly universal in patients with primary immunodeficiency diseases (PIDD). Early recognition, aggressive treatment, and prophylaxis with antimicrobials and immunoglobulin replacement have been the mainstays of management and will be reviewed here with an emphasis on respiratory infections. Genetic discoveries have allowed direct translation of research to clinical practice, improving our understanding of clinical patterns of pathogen susceptibilities and guiding prophylaxis. The recent identification of inborn errors in type I interferon signaling as a basis for life-threatening viral infections in otherwise healthy individuals suggests another targetable pathway for treatment and/or prophylaxis. The future of PIDD diagnosis will certainly involve early genetic identification by newborn screening before onset of infections, with early treatment offering the potential of preventing disease complications such as chronic lung changes. Gene editing approaches offer tremendous therapeutic potential, with rapidly emerging delivery systems. Antiviral therapies are desperately needed, and specific cellular therapies show promise in patients requiring hematopoietic stem cell transplantation. The introduction of approved therapies for clinical use in PIDD is limited by the difficulty of studying outcomes in rare patients/conditions with conventional clinical trials., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: Despite a better understanding of the epidemiology, pathogenesis, and management of patients with anaphylaxis, there remain knowledge gaps. Enumerating and prioritizing these gaps would allow limited scientific resources to be directed more effectively., Objective: We sought to systematically describe and appraise anaphylaxis knowledge gaps and future research priorities based on their potential impact and feasibility., Methods: We convened a 25-member multidisciplinary panel of anaphylaxis experts. Panelists formulated knowledge gaps/research priority statements in an anonymous electronic survey. Four anaphylaxis themed writing groups were formed to refine statements: (1) Population Science, (2) Basic and Translational Sciences, (3) Emergency Department Care/Acute Management, and (4) Long-Term Management Strategies and Prevention. Revised statements were incorporated into an anonymous electronic survey, and panelists were asked to rate the impact and feasibility of addressing statements on a continuous 0 to 100 scale., Results: The panel generated 98 statements across the 4 anaphylaxis themes: Population Science (29), Basic and Translational Sciences (27), Emergency Department Care/Acute Management (24), and Long-Term Management Strategies and Prevention (18). Median scores for impact and feasibility ranged from 50.0 to 95.0 and from 40.0 to 90.0, respectively. Key statements based on median rating for impact/feasibility included the need to refine anaphylaxis diagnostic criteria, identify reliable diagnostic, predictive, and prognostic anaphylaxis bioassays, develop clinical prediction models to standardize postanaphylaxis observation periods and hospitalization criteria, and determine immunotherapy best practices., Conclusions: We identified and systematically appraised anaphylaxis knowledge gaps and future research priorities. This study reinforces the need to harmonize scientific pursuits to optimize the outcomes of patients with and at risk of anaphylaxis., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Purpose of Review: A known history of a severe allergic reaction (e.g., anaphylaxis) to any component of the vaccine is the only contraindication to coronavirus disease 2019 (COVID-19) mRNA vaccination. It is important for pediatricians to understand the likelihood of an allergic reaction to COVID-19 mRNA vaccines, including its excipients., Recent Findings: Episodes concerning for anaphylaxis were immediately reported following early administration of COVID-19 mRNA vaccines to adults. Although allergic type symptoms were reported equally in recipients of placebos and test vaccines in phase 3 clinical trials, post-authorization prospective studies state that 0.2-2% of vaccine recipients have experienced allergic reactions. Subsequent allergy testing of affected individuals has focused largely on evaluation of allergic sensitization to a novel vaccine excipient, polyethylene glycol (PEG). PEG is a polymer incorporated in numerous pharmaceutical products because of its favorable, inert properties. The results of allergy testing in adults to date indicate that IgE mediated anaphylaxis to PEG allergy is rarely identified after COVID-19 mRNA vaccine reactions. Numerous individuals with presumed anaphylaxis have tolerated a second vaccine after evaluation and testing by an allergist, suggesting either misdiagnosis or a novel immune mechanism., Summary: Confirmed anaphylactic reactions to COVID-19 mRNA vaccines are rare, likely due to a lack of preexisting IgE against the vaccine components, including PEG., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: There is no widely adopted severity grading system for acute allergic reactions, including anaphylactic and nonanaphylactic reactions, thus limiting the ability to optimize and standardize management practices and advance research., Objective: The aim of this study was to develop a severity grading system for acute allergic reactions for use in clinical care and research., Methods: From May to September 2020, we convened a 21-member multidisciplinary panel of allergy and emergency care experts; 9 members formed a writing group to critically appraise and assess the strengths and limitations of prior severity grading systems and develop the structure and content for an optimal severity grading system. The entire study panel then revised the grading system and sought consensus by utilizing Delphi methodology., Results: The writing group recommended that an optimal grading system encompass the severity of acute allergic reactions on a continuum from mild allergic reactions to anaphylactic shock. Additionally, the severity grading system must be able to discriminate between clinically important differences in reaction severity to be relevant in research while also being intuitive and straightforward to apply in clinical care. Consensus was reached for all elements of the proposed severity grading system., Conclusion: We developed a consensus severity grading system for acute allergic reactions, including anaphylactic and nonanaphylactic reactions. Successful international validation, refinement, dissemination, and application of the grading system will improve communication among providers and patients about the severity of allergic reactions and will help advance future research., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Anaphylaxis to vaccines is historically a rare event. The coronavirus disease 2019 pandemic drove the need for rapid vaccine production applying a novel antigen delivery system: messenger RNA vaccines packaged in lipid nanoparticles. Unexpectedly, public vaccine administration led to a small number of severe allergic reactions, with resultant substantial public concern, especially within atopic individuals. We reviewed the constituents of the messenger RNA lipid nanoparticle vaccine and considered several contributors to these reactions: (1) contact system activation by nucleic acid, (2) complement recognition of the vaccine-activating allergic effector cells, (3) preexisting antibody recognition of polyethylene glycol, a lipid nanoparticle surface hydrophilic polymer, and (4) direct mast cell activation, coupled with potential genetic or environmental predispositions to hypersensitivity. Unfortunately, measurement of anti-polyethylene glycol antibodies in vitro is not clinically available, and the predictive value of skin testing to polyethylene glycol components as a coronavirus disease 2019 messenger RNA vaccine-specific anaphylaxis marker is unknown. Even less is known regarding the applicability of vaccine use for testing (in vitro/vivo) to ascertain pathogenesis or predict reactivity risk. Expedient and thorough research-based evaluation of patients who have suffered anaphylactic vaccine reactions and prospective clinical trials in putative at-risk individuals are needed to address these concerns during a public health crisis., (Copyright © 2021 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Background: The use of inconsistent definitions for anaphylaxis outcomes limits our understanding of the natural history and epidemiology of anaphylaxis, hindering clinical practice and research efforts., Objective: Our aim was to develop consensus definitions for clinically relevant anaphylaxis outcomes by utilizing a multidisciplinary group of clinical and research experts in anaphylaxis., Methods: Using Delphi methodology, we developed agenda topics and drafted questions to review during monthly conference calls. Through online surveys, a 19-member panel consisting of experts in allergy and/or immunology and emergency medicine rated their level of agreement with the appropriateness of statements on a scale of 1 to 9. A median value of 1.0 to 3.4 was considered inappropriate, a median value of 3.5 to 6.9 was considered uncertain, and a median value of 7.0 to 9.0 was considered appropriate. A disagreement index was then calculated, with values less than 1.0 categorized as "consensus reached." If consensus was not reached after the initial survey, subsequent surveys incorporating the aggregate de-identified responses from prior surveys were sent to panel members. This process was repeated until consensus was reached or 4 survey rounds had been completed, after which the question was categorized as "no consensus reached.", Results: The panel developed outcome definitions for persistent, refractory, and biphasic anaphylaxis, as well as for persistent and biphasic nonanaphylactic reactions. There was also consensus among panel members regarding the need to develop an anaphylaxis severity grading system., Conclusion: Dissemination and application of these definitions in clinical care and research will help standardize the terminology used to describe anaphylaxis outcomes and serve as the foundation for future research, including research aimed at development of an anaphylaxis severity grading system., (Copyright © 2020 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

This article explores the distribution and mutation spectrum of potential disease-causing genetic variants in hemophagocytic lymphohistiocytosis (HLH)-associated genes observed in a large tertiary clinical referral laboratory. Samples from 1892 patients submitted for HLH genetic analysis were studied between September 2013 and June 2018 using a targeted next-generation sequencing panel approach. Patients ranged in age from 1 day to 78 years. Analysis included 15 genes associated with HLH. A potentially causal genetic finding was observed in 227 (12.0%) samples in this cohort. A total of 197 patients (10.4%) had a definite genetic diagnosis. Patients with pathogenic variants in familial HLH genes tended to be diagnosed significantly younger compared with other genes. Pathogenic or likely pathogenic variants in the PRF1 gene were the most frequent. However, mutations in genes associated with degranulation defects (STXBP2, UNC13D, RAB27A, LYST, and STX11) were more common than previously appreciated and collectively represented >50% of cases. X-linked conditions (XIAP, SH2D1A, and MAGT1) accounted for 17.8% of the 197 cases. Pathogenic variants in the SLC7A7 gene were the least encountered. These results describe the largest cohort of genetic variation associated with suspected HLH in North America. Merely 10.4% of patients were identified with a clearly genetic cause by this diagnostic approach; other possible etiologies of HLH should be investigated. These results suggest that careful thought should be given regarding whether patients have a clinical phenotype most consistent with HLH vs other clinical and disease phenotypes. The gene panel identified known pathogenic and novel variants in 10 HLH-associated genes., (© 2020 by The American Society of Hematology.)

Hemophagocytic lymphohistiocytosis (HLH) is an overwhelming clinical syndrome associated with extreme immune activation. Familial HLH is caused by autosomal-recessive inheritance of gene mutations that cripple lymphocyte cytotoxicity. X-linked lymphoproliferative diseases and mutations in Nod-like receptor caspase activation and recruitment domain containing protein 4 (NLRC4) also feature HLH as a predominant manifestation. In addition, "secondary" HLH may occur in immunocompromized patients or in individuals with previously intact immune responses in the context of strong immunologic triggers such as EBV infection, malignancy, rheumatologic disease, and drug hypersensitivity. Regardless of the etiology, HLH is often fatal unless recognized and treated aggressively. Research over the last 20 years has led to many advances in diagnosis and treatment. Rapid testing strategies designed to quickly screen for immune activation and cytotoxic lymphocyte dysfunction are now clinically available and genetic panels/testing algorithms may accelerate a genetic diagnosis. Immunosuppressive treatment protocols have been refined, and experience is gaining with alternative and salvage approaches. However, these advances improve the outcome of patients only when the diagnosis of HLH is made. Ongoing education is needed to ensure medical providers can appropriately recognize and diagnose HLH. This Grand Rounds Review will summarize the clinical and diagnostic features of HLH and highlight known genetic causes., (Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Growing interest in natural killer (NK) cell-based therapy for treating human cancer has made it imperative to develop new tools to measure early events in cell death. We recently demonstrated that protease-cleavable luciferase biosensors detect granzyme B and pro-apoptotic caspase activation within minutes of target cell recognition by murine cytotoxic lymphocytes. Here we report successful adaptation of the biosensor technology to assess perforin-dependent and -independent induction of death pathways in tumor cells recognized by human NK cell lines and primary cells. Cell-cell signaling via both Fc receptors and NK-activating receptors led to measurable luciferase signal within 15 minutes. In addition to the previously described aspartase-cleavable biosensors, we report development of granzyme A and granzyme K biosensors, for which no other functional reporters are available. The strength of signaling for granzyme biosensors was dependent on perforin expression in IL-2-activated NK effectors. Perforin-independent induction of apoptotic caspases was mediated by death receptor ligation and was detectable after 45 minutes of conjugation. Evidence of both FasL and TRAIL-mediated signaling was seen after engagement of Jurkat cells by perforin-deficient human cytotoxic lymphocytes. Although K562 cells have been reported to be insensitive to TRAIL, robust activation of pro-apoptotic caspases by NK cell-derived TRAIL was detectable in K562 cells. These studies highlight the sensitivity of protease-cleaved luciferase biosensors to measure previously undetectable events in live cells in real time. Further development of caspase and granzyme biosensors will allow interrogation of additional features of granzyme activity in live cells including localization, timing, and specificity., (© 2015 by The American Society of Hematology.)

Defects in perforin lead to the failure of T and NK cell cytotoxicity, hypercytokinemia, and the immune dysregulatory condition known as familial hemophagocytic lymphohistiocytosis (FHL). The only curative treatment is allogeneic hematopoietic stem cell transplantation which carries substantial risks. We used lentiviral vectors (LV) expressing the human perforin gene, under the transcriptional control of the ubiquitous phosphoglycerate kinase promoter or a lineage-specific perforin promoter, to correct the defect in different murine models. Following LV-mediated gene transfer into progenitor cells from perforin-deficient mice, we observed perforin expression in mature T and NK cells, and there was no evidence of progenitor cell toxicity when transplanted into irradiated recipients. The resulting perforin-reconstituted NK cells showed partial recovery of cytotoxicity, and we observed full recovery of cytotoxicity in polyclonal CD8(+) T cells. Furthermore, reconstituted T cells with defined antigen specificity displayed normal cytotoxic function against peptide-loaded targets. Reconstituted CD8(+) lymphoblasts had reduced interferon-γ secretion following stimulation in vitro, suggesting restoration of normal immune regulation. Finally, upon viral challenge, mice with >30% engraftment of gene-modified cells exhibited reduction of cytokine hypersecretion and cytopenias. This study demonstrates the potential of hematopoietic stem cell gene therapy as a curative treatment for perforin-deficient FHL.

Activation of caspase-mediated apoptosis is reported to be a hallmark of both granzyme B- and Fas-mediated pathways of killing by CTLs; however, the kinetics of caspase activation remain undefined owing to an inability to monitor target cell-specific apoptosis in real time. We have overcome this limitation by developing a novel biosensor assay that detects continuous, protease-specific activity in target cells. Biosensors were engineered from a circularly permuted luciferase, linked internally by either caspase 3/7 or granzyme B/caspase 8 cleavage sites, thus allowing activation upon proteolytic cleavage by the respective proteases. Coincubation of murine CTLs with target cells expressing either type of biosensor led to a robust luminescent signal within minutes of cell contact. The signal was modulated by the strength of TCR signaling, the ratio of CTL/target cells, and the type of biosensor used. Additionally, the luciferase signal at 30 min correlated with target cell death, as measured by a (51)Cr-release assay. The rate of caspase 3/7 biosensor activation was unexpectedly rapid following granzyme B- compared with Fas-mediated signal induction in murine CTLs; the latter appeared gradually after a 90-min delay in perforin- or granzyme B-deficient CTLs. Remarkably, the Fas-dependent, caspase 3/7 biosensor signal induced by perforin-deficient human CTLs was also detectable after a 90-min delay when measured by redirected killing. Thus, we have used a novel, real-time assay to demonstrate the distinct pattern of caspase activation induced by granzyme B versus Fas in human and murine CTLs., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Patients with hemophagocytic lymphohistiocytosis (HLH) exhibit immune hyper-activation as a consequence of genetic defects in secretory granule proteins of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Murine models of HLH demonstrate significant activation of CTL as central to the disease pathogenesis, but evaluation of CTL and NK activation in children with HLH or inflammatory conditions is not well described. CD8 T cells only express granzyme B (GrB) following stimulation and differentiation into CTL; therefore, we reasoned that GrB expression may serve as a signature of CTL activation. It is unknown whether human NK cells are similarly activated in vivo, as marked by increased granule proteins. Perforin and GrB levels are measured in both CTL and NK cells by flow cytometry to diagnose perforin deficiency. We retrospectively compared GrB expression in blood samples from 130 children with clinically suspected and/or genetically defined HLH to age-matched controls. As predicted, CD8 expressing GrB cells were increased in HLH, regardless of genetic etiology. Remarkably, the GrB protein content also increased in NK cells in patients with HLH and decreased following immunosuppressive therapy. This suggests that in vivo activation of NK cells occurs in hyper-inflammatory conditions. We conclude that increased detection of GrB in CTL and NK are an immune signature for lymphocyte activation in HLH, irrespective of genetic subtype and may also be a useful measure of immune activation in other related conditions.

Familial hemophagocytic lymphohistiocytosis (HLH) is a rare primary immunodeficiency disorder characterized by defects in cell-mediated cytotoxicity that results in fever, hepatosplenomegaly, and cytopenias. Familial HLH is well recognized in children but rarely diagnosed in adults. We conducted a retrospective review of genetic and immunologic test results in patients who developed HLH in adulthood. Included in our study were 1531 patients with a clinical diagnosis of HLH; 175 patients were 18 years or older. Missense and splice-site sequence variants in PRF1, MUNC13-4, and STXBP2 were found in 25 (14%) of the adult patients. The A91V-PRF1 genotype was found in 12 of these patients (48%). The preponderance of hypomorphic mutations in familial HLH-causing genes correlates with the later-onset clinical symptoms and the more indolent course in adult patients. We conclude that late-onset familial HLH occurs more commonly than was suspected previously.

Familial hemophagocytic lymphohistiocytosis (FHL) is a rare, genetically heterogeneous autosomal recessive immune disorder that results when the critical regulatory pathways that mediate immune defense mechanisms and the natural termination of immune/inflammatory responses are disrupted or overwhelmed. To advance the understanding of FHL, we performed gene expression profiling of peripheral blood mononuclear cells from 11 children with untreated FHL. Total RNA was isolated and gene expression levels were determined using microarray analysis. Comparisons between patients with FHL and normal pediatric controls (n = 30) identified 915 down-regulated and 550 up-regulated genes with more than or equal to 2.5-fold difference in expression (P ≤ .05). The expression of genes associated with natural killer cell functions, innate and adaptive immune responses, proapoptotic proteins, and B- and T-cell differentiation were down-regulated in patients with FHL. Genes associated with the canonical pathways of interleukin-6 (IL-6), IL-10 IL-1, IL-8, TREM1, LXR/RXR activation, and PPAR signaling and genes encoding of antiapoptotic proteins were overexpressed in patients with FHL. This first study of genome-wide expression profiling in children with FHL demonstrates the complexity of gene expression patterns, which underlie the immunobiology of FHL.

Background: Deficiency of X-linked inhibitor of apoptosis (XIAP), caused by BIRC4 gene mutations, is the second known cause of X-linked lymphoproliferative disease (XLP), a rare primary immunodeficiency that often presents with life-threatening hemophagocytic lymphohistiocytosis (HLH). Rapid diagnosis of the known genetic causes of HLH, including XIAP deficiency, facilitates the initiation of life-saving treatment and preparation for allogeneic hematopoietic cell transplantation (HCT). Until now, a rapid screening test for XIAP deficiency has not been available., Methods: To develop a flow cytometric screening test for XIAP deficiency, we first used lymphoblastic cell lines generated from controls and patients with BIRC4 mutations to identify two commercially available antibodies specific for native intracellular XIAP. Next, we used these antibodies to study control whole blood leukocyte XIAP expression. We then studied XIAP expression in leukocytes from patients with XLP due to BIRC4 mutations, maternal carriers, and patients following HCT., Results: XIAP was expressed by the majority of all whole blood nucleated cells in normal controls. In contrast, XIAP was absent or decreased in all lymphocyte subsets, monocytes and granulocytes from four unrelated patients with XLP due to BIRC4 mutations. Bimodal distribution of XIAP expression was evident in two maternal carriers, with significant skewing toward cells expressing normal XIAP. Bimodal distribution was also observed in a patient following HCT., Conclusions: Flow cytometric analysis of intracellular XIAP provides a rapid screening test for XLP due to XIAP deficiency. It also allows carrier detection and can be used to monitor donor versus recipient reconstitution following HCT., ((c) 2009 Clinical Cytometry Society.)

Human invariant natural killer T cells (iNKT cells) are a unique population of T cells that express an invariantly rearranged T-cell receptor (TCR) composed of TCRValpha24 and TCRVbeta11 chains which recognize glycosphingolipid antigens presented by the CD1d molecule. iNKT cells are absent in patients with X-linked lymphoproliferative disease (XLP) due to SH2D1A mutation, and are reported to be decreased in patients with XLP due to BIRC4 mutations. However, mice deficient in the BIRC4 gene product, X-linked Inhibitor of Apoptosis (XIAP), have normal iNKT cell populations. Because of this, we studied iNKT cell populations in 6 patients with XLP due to BIRC4 mutations, with comparison to 103 pediatric and adult normal control samples. We found that iNKT cells constitute 0.008%-1.176% of normal peripheral blood T cells, and that iNKT cell populations were normal or increased in patients with BIRC4 mutations. We conclude that XLP due to BIRC4 mutation is not associated with decreased populations of iNKT cells, and that XIAP is likely not a requirement for iNKT cell development.

Perforin-mediated lymphocyte cytotoxicity is critical for pathogen elimination and immune homeostasis. Perforin disruption of target cell membranes is hypothesized to require binding of a calcium-dependent, lipid-inserting, C2 domain. In a family affected by hemophagocytic lymphohistiocytosis, a severe inflammatory disorder caused by perforin deficiency, we identified 2 amino acid substitutions in the perforin C2 domain: T435M, a previously identified mutant with disputed pathogenicity, and Y438C, a novel substitution. Using biophysical modeling, we predicted that the T435M substitution, but not Y438C, would interfere with calcium binding and thus cytotoxic function. The capacity for cytotoxic function was tested after expression of the variant perforins in rat basophilic leukemia cells and murine cytotoxic T lymphocytes. As predicted, cells transduced with perforin-T435M lacked cytotoxicity, but those expressing perforin-Y438C displayed intact cytotoxic function. Using novel antibody-capture and liposome-binding assays, we found that both mutant perforins were secreted; however, only nonmutated and Y438C-substituted perforins were capable of calcium-dependent lipid binding. In addition, we found that perforin-Y438C was capable of mediating cytotoxicity without apparent proteolytic maturation. This study clearly demonstrates the pathogenicity of the T435M mutation and illustrates, for the first time, the critical role of the human perforin C2 domain for calcium-dependent, cytotoxic function.

Background: Pediatric eosinophilic esophagitis (EE) is a recently described disorder associated with atopy. Although studies of esophageal tissue suggest that Th2 cytokines and eotaxin-3 may be crucial in disease pathogenesis, little is known about the systemic immunological phenotypes of children with EE., Objectives: To define the phenotypes of peripheral blood eosinophils and lymphocytes in EE and to examine for correlations between these parameters and tissue eosinophil numbers and disease severity., Patients and Methods: Blood was collected from children with EE, atopic control children without EE, and nonatopic control children without EE. Flow cytometry was used to measure eosinophil expression of chemokine receptor 3 (CCR3) and interleukin-5 receptor-alpha (IL-5Ralpha), and intracellular lymphocyte expression of IL-4, IL-5, IL-13, interferon-gamma, and tumor necrosis factor-alpha. Eosinophil numbers and eotaxin-3 mRNA levels were quantitated in esophageal biopsy specimens., Results: Compared with nonatopic control children, EE patients with active disease had increased peripheral blood eosinophil percentages, mean channel of fluorescence (MCF) of CCR3 on eosinophils, and percentage of CD4+ T cells expressing IL-5. Notably, these parameters positively correlated with esophageal eosinophil numbers. Eotaxin-3 tissue expression positively correlated with esophageal eosinophil numbers and peripheral blood eosinophil CCR3 MCF. The percentage of peripheral blood eosinophils, eosinophil CCR3 MCF, and CD4+ T cell expression of IL-5 were lower in EE patients in disease remission than in patients with active disease., Conclusions: Collectively, these studies demonstrate cooperation between systemic CD4+ Th2-cell-mediated immunity and an enhanced eosinophil-CCR3/eotaxin-3 pathway in EE pathogenesis. Furthermore, the imbalanced Th2 immunity and increased CCR3 expression are reversible with disease remission.

Background: Childhood interstitial lung disease (ILD) is a spectrum of diseases including many different rare lung conditions. We present a family with an unusual presentation of ILD in association with rheumatologic and immunologic abnormalities., Methods: Eight children with a common father were evaluated for evidence of lung disease in association with rheumatologic findings. All underwent routine history and physical examination, hematologic evaluation, and chest radiography and/or CT scan of the chest. Seven children underwent a more extensive immunologic evaluation. Those who were able underwent pulmonary function testing, and four children underwent lung biopsy., Results: Six of eight children with a common father were found to have radiographic findings consistent with ILD. These children also had evidence of autoimmune disease with joint symptoms, alopecia, rheumatoid factor production, and hypergammaglobulinemia. Open-lung biopsy in four children revealed a spectrum of pulmonary lymphoid proliferations ranging from reactive lymphoid hyperplasia to lymphoid interstitial pneumonia., Conclusion: The findings of ILD and autoimmunity in a kindred of children suggest a novel genetic disorder of autosomal dominant pattern and variable penetrance. Although the precise pathogenesis remains unclear, these cases provide valuable insight into childhood ILD.

Missense mutations in perforin, a critical effector of lymphocyte cytotoxicity, lead to a spectrum of diseases, from familial hemophagocytic lymphohistiocytosis to an increased risk of tumorigenesis. Understanding of the impact of mutations has been limited by an inability to express human perforin in vitro. We have shown, for the first time to our knowledge, that recombinant human perforin is expressed, processed appropriately, and functional in rat basophilic leukemia (RBL) cells following retroviral transduction. Subsequently, we have addressed how perforin missense mutations lead to absent perforin detection and impaired cytotoxicity by analyzing 21 missense mutations by flow cytometry, immunohistochemistry, and immunoblot. We identified perforin missense mutations with partial maturation (class 1), no apparent proteolytic maturation (class 2), and no recognizable forms of perforin (class 3). Class 1 mutations exhibit lytic function when expressed in RBL cells and are associated with residual protein detection and variable cytotoxic function in affected individuals, suggesting that carriers of class 1 alleles may exhibit more subtle immune defects. By contrast, class 3 mutations cause severely diminished perforin detection and cytotoxicity, while class 2 mutations have an intermediate phenotype. Thus, the pathologic mechanism of perforin missense mutation likely involves a protein dosage effect of the mature protein.

In this article we describe a clinical entity appearing in seven preadolescent patients who presented with chronic red papules within a prominent nasal crease. Milia were also noted in the nasal crease, but there was no evidence of acne vulgaris. The duration of symptoms was 4 months to 2 years, and lesions ranged from inflamed red papules, which were treated with topical antiinflammatory medications, to scarred white papules requiring excision. Histologic evaluation of two lesions revealed keratin granulomas that were likely derived from ruptured, inflamed milia. Due to its similarity in appearance to acne vulgaris, but different pathogenesis and clinical course, we suggest naming this newly described entity "pseudoacne of the nasal crease."

Asthma is a complex polygenic disease. Many studies have implicated the importance of IL-4R alpha in the development of allergic inflammation and its gene has been implicated in the genetics of asthma and atopy. In this study, we examined the functional consequences of two of the human IL-4R alpha allelic variants that have been found to associate with asthma and atopy. We examined the effects of each variant alone and in combination on IL-4-dependent gene induction. We found that neither the Q576R nor the I75V variants affected IL-4-dependent CD23 expression. However, the combination of V75R576 resulted in expression of an IL-4R alpha with enhanced sensitivity to IL-4. We next examined the genetics of five of the known IL-4R alpha allelic variants in asthmatic and nonatopic populations. Strikingly, the association of V75/R576 with atopic asthma was greater than either allele alone and the association of R576 with atopic asthma was dependent on the coexistence of V75. A haplotype analysis revealed a single IL-4R alpha haplotype that was associated with allergic asthma, VACRS, further confirming the importance of the V75 and R576 combination in the genetics of asthma. This is the first report demonstrating that a functional alteration in IL-4R alpha requires the coexistence of two naturally occurring single nucleotide polymorphisms (snps) in combination; neither snp alone is sufficient. These data illustrate the importance of studying snps in combination, because the functional significance of a given snp may only be evident in a specific setting of additional snps in the same or different genes.

In women, chronically elevated androgens have been associated with polycystic ovarian syndrome and infertility. Recently, we described transgenic mice with elevated serum LH secondary to targeted expression of a transgene encoding a chimeric LH beta-subunit. Mature transgenic females exhibit elevated androgens, anovulation, and a range of ovarian phenotypes including cysts, widespread luteinization, and tumors. In the present study we have examined serum levels of LH and testosterone and the concurrent development of the reproductive system in prepubertal mice. Serum LH in prepubertal females was elevated despite increased serum testosterone and estradiol, indicating a relative insensitivity to steroid negative feedback. Elevated serum LH and hyperandrogenemia resulted in accelerated vaginal opening and ovarian follicular development in transgenic females. Precocious antral follicle formation and conspicuous hypertrophy of the theca-interstitium preceded the development of large cysts with marked hemorrhage. Based on these studies we conclude that chronic prepubertal elevation of serum LH results in gonadotropin-dependent hyperandrogenemia, leading to abnormal sexual development and significant ovarian pathology.

The intracellular hydrophilic region of the cystic fibrosis transmembrane conductance regulator (CFTR), the R domain, has been postulated to be a regulator of the Cl-channel. Under basal conditions R blocks the channel, but when phosphorylated, R undergoes conformational change to open the channel. Overexpression of R in 9/HTEo- cells, a human tracheal epithelial cell line with adenosine 3',5' -cyclic monophosphate (cAMP)-regulated Cl- conductance due to CFTR, caused reduced basal Cl- conductance and elimination of its response to isoproterenol, but ionomycin-stimulated Cl- efflux was preserved. Cells which overexpressed R showed no downregulation of endogenous CFTR mRNA and had normal cAMP production and protein kinase A (PKA) activity, so R did not act at these levels. Although the precise mechanism by which R affects CFTR conductance is undetermined, these cell lines could be useful in separating the cell biological consequences of impaired Cl- transport from those of mutant CFTR per se.

Hypersecretion of luteinizing hormone (LH) is implicated in infertility and miscarriages in women. A lack of animal models has limited progress in determining the mechanisms of LH toxicity. We have recently generated transgenic mice expressing a chimeric LH beta subunit (LH beta) in gonadotropes. The LH beta chimera contains the C-terminal peptide of the human chorionic gonadotropin beta subunit. Addition of this peptide to bovine LH beta resulted in a hormone with a longer half-life. Furthermore, targeted expression of the LH beta chimera led to elevated LH levels and infertility in female transgenics. These mice ovulated infrequently, maintained a prolonged luteal phase, and developed pathologic ovarian changes such as cyst formation, marked enlargement of ovaries, and granulosa cell tumors. Testosterone and estradiol levels were increased compared to nontransgenic littermates. An unusual extragonadal phenotype was also observed: transgenic females developed hydronephropathy and pyelonephritis. The pathology observed demonstrates a direct association between abnormal secretion of LH and infertility and underscores the utility of the transgenic model for studying how excess LH leads to cyst formation, ovarian tumorigenesis, and infertility.