A cell-based functional assay that accurately links genotype to phenotype in familial HLH.

Familial forms of the severe immunoregulatory disease hemophagocytic lymphohistiocytosis (HLH) arise from biallelic mutations in the PRF1, UNC13D, STXBP2, and STX11 genes. Early and accurate diagnosis of the disease is important to determine the most appropriate treatment option, including potentially curative stem cell transplantation. The diagnosis of familial HLH (FHL) is traditionally based on finding biallelic mutations in patients with HLH symptoms and reduced natural killer (NK)-cell cytotoxicity. However, patients often have a low NK-cell count or receive immunosuppressive therapies that may render the NK-cell cytotoxicity assay unreliable. Furthermore, to fully understand the nature of a disease it is critical to directly assess the effect of mutations on cellular function; this will help to avoid instances in which carriers of innocuous mutations may be recommended for invasive procedures including transplantation. To overcome this diagnostic problem, we have developed a rapid and robust method that takes advantage of the functional equivalence of the human and mouse orthologues of PRF1, UNC13D, STX11, and STXBP2 proteins. By knocking out endogenous mouse genes in CD8+ T cells and simultaneously replacing them with their mutated human orthologues, we can accurately assess the effect of mutations on cell function. The wide dynamic range of this novel system allowed us to understand the basis of, otherwise cryptic, cases of FHL or HLH and, in some instances, to demonstrate that previously reported mutations are unlikely to cause FHL. This novel approach provides valuable new information to enable more accurate diagnosis and treatment of patients with HLH or FHL who inherit mutations of undetermined pathogenicity.

Utility of Follow-up Radiographs in Type 1 Supracondylar Humerus Fractures.

OBJECTIVE: Supracondylar humerus (SCH) fractures are common among pediatric patients, with the severity categorized using the Gartland classification system. Type 1 SCH fractures are nondisplaced and treated with immobilization, while more displaced fractures require surgery. The need for follow-up radiographs, particularly for type 1 fractures, is an area where evidence is lacking. This study investigates the clinical value and financial implications of follow-up radiographs for type 1 SCH fractures, hypothesizing that they do not alter clinical management and, therefore, represent an unnecessary expense. METHODS: This retrospective cohort study, approved by the Institutional Review Board, focused on patients under 18 with nondisplaced SCH fractures treated nonoperatively. One hundred one type 1 SCH fractures, in which the fracture was visible on presenting radiographs, were chosen from patients presenting between January 2021 and December 2022. Charts were reviewed for demographic information, time of cast removal, and complications. A pediatric orthopaedic surgeon and orthopaedic resident reviewed the radiographs to confirm the injury to be a type 1 SCH fracture. RESULTS: Among the 101 patients, after the initial presentation, 79 attended an interim visit and 101 attended a "3-week follow-up" at an average of 23 days postinjury. All patients underwent radiographs during these visits for a total of 180 radiographs after confirmation of type 1 SCH fracture. No changes in management resulted from follow-up radiographs. One instance of refracture was noted ~3 months after cast removal. There were 180 superfluous follow-up radiographs taken at subsequent clinic visits. The total charge for these radiographs was $76,001.40, averaging $752.49 per patient. CONCLUSION: Follow-up radiographs for type 1 SCH fractures did not lead to changes in clinical management, aligning with previous findings in more severe SCH fractures. This approach can reduce costs, radiation exposure, and clinic time without compromising patient care. The study can reassure providers and parents about the lack of necessity for follow-up radiographs to document healing. LEVEL OF EVIDENCE: Level-IV.

Understanding the ongoing learning needs of Australian paediatricians: Evaluation of a pilot paediatric video teaching programme.

AIM: The purpose of this study was to evaluate whether pre-recorded video-based lectures (VBLs) covering a range of paediatric topics are an acceptable means of providing ongoing education for consultant and trainee paediatricians in Australia. METHODS: Previous participants (paediatric consultants and junior medical officers) of a neurology outreach teleconference programme offered by a paediatric neurologist between 2017 and 2020 were invited to participate in a multi-specialty pre-recorded video-based education programme. Acceptability was explored by assessing relevance, likelihood of utilising VBL's in the future, uptake and learning activity preferences. The impact of VBLs on confidence, currency and practice was also explored. Additional data including topics of interest, preferred video format, duration, viewing method and frequency of delivery were captured, to better understand participant preferences to inform future efforts. RESULTS: A total of 135 consented; 116 returned baseline; 94 returned follow-up surveys. Preferred learning activities included a live/interactive component. Videos were considered relevant. Preferences for pre-recorded videos improved from ninth to sixth most preferred learning activity post-intervention. VBL convenience and accessibility were valued. Practice was altered in: approach to management, use of treatments, confidence in decision-making, and discussion with families and patients. The average view duration was 16 min. Longer videos yielded slightly lower audience retention rates. For future offerings, the majority endorsed a preference for a 'mixed' video format and duration of 20-40 min, offered monthly. CONCLUSION: Video-based medical education is an appealing and sustainable alternative, given the convenience of unrestricted accessibility, in meeting ongoing learning needs of Australian paediatricians and trainees.

The FOXP3 full-length isoform controls the lineage-stability of CD4+FOXP3+ regulatory T cells.

The transcription factor FOXP3 is essential for CD4+FOXP3+ regulatory T (Treg) cell development and function. Human FOXP3 exists in distinct isoforms and alterations in isoform expression is associated with inflammatory disease progression, however, the exact functions of FOXP3 isoforms remain poorly understood. Herein we used flow cytometry and RNA-sequencing to analyze subsets of Treg cells from two IPEX patients, and a healthy carrier, of a recently described FOXP3 mutation (c.305delT). This mutation is located in exon 2 and results in the loss of the full-length FOXP3 isoform. Treg cells lacking full-length FOXP3 are found at lower-than-expected frequencies. This loss cannot be explained solely by altered thymic output, changes in proliferation, peripheral induction of Treg cells, or apoptosis. Instead, fulllength FOXP3 control a distinct genetic program, involving the previously identified FOXP3 regulators ID3, BCL6 and eIF4E, that upholds Treg cell lineage stability, while it appears nonessential for Treg cell activation.

ESCAPE-Allergy: Evaluating screening for children and adolescents with penicillin allergy.

AIM: Penicillin allergy labels are frequently encountered in children and are associated with significant harms. Most children are falsely labelled and can safely tolerate a penicillin but delabelling strategies are underutilised and paediatric-specific resources are lacking. The aim of this study was to evaluate an allergy assessment tool for children in hospital. METHODS: We evaluated a paediatric-adapted penicillin allergy assessment tool, using an online survey of clinicians in a tertiary paediatric hospital, with 10 hypothetical potential penicillin allergy or adverse reaction cases (including non-allergy reactions). For each case, respondents were asked to use the tool to assign a reaction phenotype and recommend management. We determined the tool's sensitivity, specificity and acceptability to end users. RESULTS: We evaluated 30 complete survey responses from senior and junior medical staff, nurses and pharmacists. The tool's overall sensitivity was 80.7% (95% confidence interval (CI) 74.2-87.1%) for assigning the correct reaction phenotype and 85.3% (95% CI 79.4-91.3%) for appropriate management. The tool had high sensitivity for identifying immediate hypersensitivity reactions at 95.6% (95% CI 90.2-100%). Most respondents agreed or strongly agreed that they would use the tool in their practice (22/30, 73.3%). CONCLUSION: This survey evaluated a paediatric-adapted penicillin allergy assessment tool in a tertiary paediatric hospital among multidisciplinary clinician groups. The tool performed well overall and had high safety in identifying immediate hypersensitivity reactions. Further research to support implementation of allergy assessment and delabelling programmes among children is required.

The Role of ZEB2 in Human CD8 T Lymphocytes: Clinical and Cellular Immune Profiling in Mowat-Wilson Syndrome.

The Zeb2 gene encodes a transcription factor (ZEB2) that acts as an important immune mediator in mice, where it is expressed in early-activated effector CD8 T cells, and limits effector differentiation. Zeb2 homozygous knockout mice have deficits in CD8 T cells and NK cells. Mowat-Wilson syndrome (MWS) is a rare genetic disease resulting from heterozygous mutations in ZEB2 causing disease by haploinsufficiency. Whether ZEB2 exhibits similar expression patterns in human CD8 T cells is unknown, and MWS patients have not been comprehensively studied to identify changes in CD8 lymphocytes and NK cells, or manifestations of immunodeficiency. By using transcriptomic assessment, we demonstrated that ZEB2 is expressed in early-activated effector CD8 T cells of healthy human volunteers following vaccinia inoculation and found evidence of a role for TGFß-1/SMAD signaling in these cells. A broad immunological assessment of six genetically diagnosed MWS patients identified two patients with a history of recurrent sinopulmonary infections, one of whom had recurrent oral candidiasis, one with lymphopenia, two with thrombocytopenia and three with detectable anti-nuclear antibodies. Immunoglobulin levels, including functional antibody responses to protein and polysaccharide vaccination, were normal. The MWS patients had a significantly lower CD8 T cell subset as % of lymphocytes, compared to healthy controls (median 16.4% vs. 25%, p = 0.0048), and resulting increased CD4:CD8 ratio (2.6 vs. 1.8; p = 0.038). CD8 T cells responded normally to mitogen stimulation in vitro and memory CD8 T cells exhibited normal proportions of subsets with important tissue-specific homing markers and cytotoxic effector molecules. There was a trend towards a decrease in the CD8 T effector memory subset (3.3% vs. 5.9%; p = 0.19). NK cell subsets were normal. This is the first evidence that ZEB2 is expressed in early-activated human effector CD8 T cells, and that haploinsufficiency of ZEB2 in MWS patients had a slight effect on immune function, skewing T cells away from CD8 differentiation. To date there is insufficient evidence to support an immunodeficiency occurring in MWS patients.

Updated anaphylaxis guidelines: management in infants and children.

Severe allergic reactions (anaphylaxis) are unpredictable, and initial signs of what could be fatal anaphylaxis can be mild Adrenaline (epinephrine) remains the first-line drug of choice for the acute management of anaphylaxis and should be administered early There are no contraindications to intramuscular adrenaline in the treatment of anaphylaxis Correct positioning of the patient is vital as death can occur within minutes if a patient stands, walks or sits up suddenly. Position the patient correctly first and then promptly administer intramuscular adrenaline Updated guidelines by the Australasian Society of Clinical Immunology and Allergy now recommend that the 0.15 mg adrenaline injector device may be prescribed for infants and children weighing 7.5-10 kg. The recommendation to use the 0.3 mg adrenaline injector device for those over 20 kg remains unchanged The adrenaline doses in Australian Prescriber's anaphylaxis wallchart remain valid.

Current perspectives on peanut allergy.

Peanut allergy is increasingly prevalent and for most patients is a life-long condition, with the potential to cause life-threatening reactions. Accurate diagnosis and appropriate management are essential to minimise risks due to accidental peanut exposure. Current management strategies focus on strict allergen avoidance and access to emergency medicines to treat potential reactions; however, active approaches are an area of intense research. Promising new methods of food allergen immunotherapy are set to change the approach to managing peanut allergic patients in the near future.

Food protein-induced enterocolitis syndrome in Australia: A population-based study, 2012-2014.

BACKGROUND: Food protein-induced enterocolitis syndrome (FPIES) is a non-IgE-mediated gastrointestinal allergic disorder. Large population-based FPIES studies are lacking. OBJECTIVE: We sought to determine the incidence and clinical characteristics of FPIES in Australian infants. METHODS: An Australia-wide survey (2012-2014) was undertaken through the Australian Paediatric Surveillance Unit, with monthly notification of new cases of acute FPIES in infants aged less than 24 months by 1400 participating pediatricians. RESULTS: Two hundred thirty infants with FPIES were identified. The incidence of FPIES in Australian infants (<24 months) was 15.4/100,000/y. Median age of first episode, diagnosis, and notification were 5, 7, and 10 months, respectively. There was no sex predilection. Seven percent of infants had siblings with a history of FPIES, and 5% reacted during exclusive breast-feeding. Sixty-eight had a single food trigger (20% had 2 and 12% had ≥3 food triggers). The most common FPIES triggers were rice (45%), cow's milk (33%), and egg (12%). Fifty-one percent of infants reacted on their first known exposure. Infants with FPIES to multiple versus single food groups were younger at the initial episode (4.6 vs 5.8 months [mean], P = .001) and more frequently had FPIES to fruits, vegetables, or both (66% vs 21%, P < .0001). Infants exclusively breast-fed for more than 4 months had a trend toward lower rates of FPIES to multiple food groups (23% vs 36%, P = .06). Sixty-four percent of infants with FPIES to multiple foods, which included cow's milk, had coassociated FPIES to solid foods. Forty-two percent of infants with FPIES to fish reacted to other food groups. CONCLUSIONS: FPIES is not rare, with an estimated incidence of 15.4/100,000/y. Rice is the most common food trigger in Australia. Factors associated with FPIES to multiple foods included early-onset disease and FPIES to fruits, vegetables, or both.

Haematopoietic stem cell transplantation for severe combined immunodeficiency: Long-term health outcomes and patient perspectives.

AIM: To examine the long-term follow-up and health outcomes of patients who have undergone haematopoietic stem cell transplant (HSCT) for severe combined immunodeficiency (SCID). METHODS: Through a structured questionnaire, we examined follow-up arrangements and long-term health outcomes in 22 children who have had a successful HSCT for SCID during the period of 1984-2012 at the Sydney Children's Hospital, Sydney, Australia. RESULTS: Most children considered themselves healthy and 'cured' from SCID. Whilst many children enjoy relatively good bio-social health outcomes, specific negative health outcomes and absenteeism from school were perceived negatively. Two-thirds of children see their general practitioner or specialist regularly; however, there did not appear to be consistency with the nature of this follow-up. CONCLUSION: The findings from our study highlight the complex bio-psychosocial health needs of post-HSCT SCID children and encourage SCID centres to consider a multidisciplinary approach to their follow-up. Further studies into the determinants of patients' perceptions of their health are needed.

Haematopoietic stem cell transplantation for primary immunodeficiency syndromes: A 5-year single-centre experience.

AIM: Haematopoietic stem cell transplantation (HSCT) is a central therapy in the treatment of primary immunodeficiency diseases (PIDs). Over the past 5 years, outcomes have been greatly improved due to earlier diagnosis, improved donor availability, advancements in graft manipulation and the use of less toxic preparative regimens. We present a 5-year audit of HSCT for PID at a single Australian tertiary hospital. METHODS: Retrospective case note review identified diagnosis, pre-transplant medical morbidity, transplant protocol, engraftment, adverse events, post-transplant immune reconstitution and general health. RESULTS: A total of 22 patients with PID underwent 24 HSCTs at our institution between 2012 and 2016. The most common indications were severe combined immunodeficiency, chronic granulomatous disease and familial haemophagocytic lymphohistiocytosis, with a genetic diagnosis in all but two patients. Reduced intensity or reduced toxicity conditioning was used in 91% of cases, and 75% of the donors were unrelated. Transplant-related mortality at day +100 was 9.5%, and cumulative overall survival was 86%. There were three mortalities, all secondary to viral infection, one of which occurred in the context of graft failure. Two patients remained on immune support, with the remainder achieving adequate immune reconstitution. CONCLUSIONS: The outcomes for HSCT for PIDs performed at Sydney Children's Hospital were in line with the world's best practice. HSCT should be considered a potential therapeutic option for all Australian PID patients with a valid disease indication.

Cerebral Vasculitis in X-linked Lymphoproliferative Disease Cured by Matched Unrelated Cord Blood Transplant.

UNLABELLED: Vasculitis occurs rarely in association with X-linked lymphoproliferative disease (XLP). There are four published cases of non-EBV XLP-associated cerebral vasculitis reported, none of whom have survived without major cognitive impairment. CASE: A 9-year old boy initially presented aged 5 years with a restrictive joint disease. He subsequently developed dysgammaglobulinemia, episodic severe pneumonitis, aplastic anaemia, gastritis and cerebral vasculitis. A diagnosis of XLP was made, based on flow cytometric analysis and the identification of a novel mutation in SH2D1A, c.96G>C. No peripheral blood lymphocyte clonal proliferation was identified and he was EBV negative, although human herpes virus-7 (HHV7) was detected repeatedly in his cerebrospinal fluid. He underwent a reduced intensity unrelated umbilical cord blood transplant, but failed to engraft. A second 5/6 matched cord gave 100 % donor engraftment. Complications included BK virus-associated haemorrhagic cystitis, a possible NK-cell mediated immune reconstitution syndrome and post-transplant anti-glomerular basement membrane disease, the latter treated with cyclophosphamide and rituximab. At +450 days post-transplant he is in remission from his vasculitis and anti-glomerular basement membrane disease, and HHV-7 has remained undetectable. CONCLUSION: This is the second published description of joint disease in XLP, and only the fourth case of non-EBV associated cerebral vasculitis in XLP, as well as being the first to be successfully treated for this manifestation. This case raises specific questions about vasculitis in XLP, in particular the potential relevance of HHV-7 to the pathogenesis.

Pediatric Supracondylar Process Fracture: Radiographic Distinction, Misdiagnosis, and Conservative Management.

A supracondylar process is an embryonic vestigial remnant of the supracondylar foramen. It was intially described as a bone spur of the distal anteromedial humerus. An 11-year-old patient with a history of an asymptomatic mass was initially misdiagnosed with osteochondroma during a visit when five years old. Radiographic analysis revealed a tapered bony prominence with lucency at the base, representing a supracondylar process fracture after a fall. He was treated with a long-arm cast with uneventful healing and symptom resolution. This case emphasizes the importance of understanding the radiographic differences between the supracondylar process and osteochondroma to prevent misdiagnosis and unnecessary evaluations. These findings suggest that conservative management without neurovascular compromise is acceptable in selected cases.

Streptococcus pyogenes flexor tenosynovitis.

We describe a case of Group A streptococcal infection originally presenting as flexor tenosynovitis. Group A Streptococcus outbreaks have been recently described in developed countries, which constitutes an epidemiological shift that hand surgeons should be aware of.

Zeb2 drives the formation of CD11c+ atypical B cells to sustain germinal centers that control persistent infection.

CD11c+ atypical B cells (ABCs) are an alternative memory B cell lineage associated with immunization, infection, and autoimmunity. However, the factors that drive the transcriptional program of ABCs have not been identified, and the function of this population remains incompletely understood. Here, we identified candidate transcription factors associated with the ABC population based on a human tonsillar B cell single-cell dataset. We identified CD11c+ B cells in mice with a similar transcriptomic signature to human ABCs, and using an optimized CRISPR-Cas9 knockdown screen, we observed that loss of zinc finger E-box binding homeobox 2 (Zeb2) impaired ABC formation. Furthermore, ZEB2 haplo-insufficient Mowat-Wilson syndrome (MWS) patients have decreased circulating ABCs in the blood. In Cd23Cre/+Zeb2fl/fl mice with impaired ABC formation, ABCs were dispensable for efficient humoral responses after Plasmodium sporozoite immunization but were required to control recrudescent blood-stage malaria. Immune phenotyping revealed that ABCs drive optimal T follicular helper (TFH) cell formation and germinal center (GC) responses and they reside at the red/white pulp border, likely permitting better access to pathogen antigens for presentation. Collectively, our study shows that ABC formation is dependent on Zeb2, and these cells can limit recrudescent infection by sustaining GC reactions.

Acute management of anaphylaxis in pregnancy.

BACKGROUND: Anaphylaxis in pregnancy is rare but can potentially be associated with significant morbidity and mortality for the mother, fetus and neonate. With appropriate and timely management, even severe anaphylaxis can be managed with excellent maternal and fetal outcomes. OBJECTIVE: The aim of this article is to provide an illustrative case and highlight current recommendations for diagnosis and management of acute maternal anaphylaxis, which have recently been reviewed and developed into a guideline by the Australasian Society of Clinical Immunology and Allergy. DISCUSSION: An understanding of management of anaphylaxis in pregnancy is essential knowledge in the general practice setting. The recommended dosage and administration of adrenaline (epinephrine) for anaphylaxis is the same in pregnant and non-pregnant patients: 0.5 mg adrenaline intramuscularly in the mid-outer thigh (or dose of 0.01 mg/kg if <50 kg). The use of adrenaline in maternal anaphylaxis is supported by various international guidelines.

Molecular Analysis of Goodpasture's Disease Following Hematopoietic Stem Cell Transplant in a Pediatric Patient, Recalls the Conformeropathy of Wild-Type Anti-GBM Disease.

Background: Goodpasture's disease (GP) is mediated by autoantibodies that bind the glomerular and alveolar basement membrane, causing rapidly progressive glomerulonephritis with or without pulmonary hemorrhage. The autoantibodies bind neoepitopes formed upon disruption of the quaternary structure of α345NC1 hexamer, a critical structural domain of α345 collagen IV scaffolds. Hexamer disruption leads to a conformational changes that transitions α3 and α5NC1 subunits into immunogens, however, the trigger remains unknown. This contrasts with another anti-GBM disease, Alports' post-transplant nephritis (APTN), where the pathogenic alloantibody binds directly to native NC1 hexamer. The current report includes the first study of antigenic specificity and allo-incompatability in anti-GBM disease occurring after allogeneic haematopoietic stem cell transplant (HSCT). Results: The anti-GBM antibodies were found to be directed predominantly against the EA epitope of the α3 NC1 monomer of collagen IV and developed rapidly in patient serum reaching peak level within 5 weeks. Autoantibody binding to native α345NC1 hexamer was minimal; however, binding was greatly increased upon dissociation of the native hexamer. There were no polymorphic genetic differences between donor and recipient collagen IV genes which would be predicted to cause a significant NC1 conformational change or to provide a target for antibody binding. Both patient and donor possessed the Goodpasture's susceptibility HLA-allele DRB1*1501. Conclusions: The current report includes the first in-depth study of allo-incompatability and antigenic specificity in anti-GBM disease occurring after allogeneic haematopoietic stem cell transplant (HSCT). No polymorphic genetic differences were identified between donor and recipient collagen IV genes which would be predicted to provide a target for antibody binding. Furthermore, autoantibody binding to native α345NC1 hexamer was minimal, increasing greatly upon dissociation of the native hexamer, resembling wild-type GP diseases and marking this as the first example of a post-HSCT conformeropathy.