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Summary Genetic testing has increased the number of variants identified in disease genes, but the diagnostic utility is limited by lack of understanding variant function. CARD11 encodes an adaptor protein that expresses dominant-negative and gain-of-function variants associated with distinct immunodeficiencies. Here, we used a “cloning-free” saturation genome editing approach in a diploid cell line to simultaneously score 2,542 variants for decreased or increased function in the region of CARD11 associated with immunodeficiency. We also described an exon-skipping mechanism for CARD11 dominant-negative activity. The classification of reported clinical variants was sensitive (94.6%) and specific (88.9%), which rendered the data immediately useful for interpretation of seven coding and splicing variants implicated in immunodeficiency found in our clinic. This approach is generalizable for variant interpretation in many other clinically actionable genes, in any relevant cell type.

Background Congenital chylothorax (CC) can have devastating consequences for neonates. We sought to determine the outcomes of cases treated at our institution and evaluate the role of fetal intervention. Materials and methods With Institutional Review Board approval, patients treated at our institution 09/2006-04/2016 with CC were reviewed. History and outcomes were compared between patients undergoing fetal intervention (fetal group) and patients who did not (control group). Results Twenty-three patients were identified. Mean gestational age at birth was 35 wk. Overall mortality was 30% (7 patients). Nineteen patients (83%) were prenatally diagnosed, and 10 patients (43%) underwent fetal intervention. Birth weight was significantly lower in the fetal group compared to the control group (median interquartile range [IQR]; 2.5 [2.3-3.0] versus 3.3 [2.6-3.7] kg, P = 0.02). Apgar scores were significantly higher in the fetal group than the control group at 1 and 5 min (median [IQR]; 6 [4-8] versus 1 [1-2], P = 0.005 and 8 [7-9] versus 2 [2-6], P = 0.008, respectively). For those patients with prenatal diagnosis of CC and hydrops fetalis, thrombosis and lymphopenia were both improved in the fetal group (thrombosis 0% versus 40%, P = 0.03; lymphocyte nadir [median {IQR}] 1.5 [0.6-2.9] versus 0.1 [0.05-0.2], P = 0.02). Duration of support with mechanical ventilation was significantly shorter in the fetal group (median [IQR]; 1 [0-40] versus 41 [29-75] d, P = 0.04). Conclusions Fetal intervention for CC is associated with improved Apgar scores and decreased ventilator days and complications in patients with hydrops fetalis. Fetuses with chylothorax, especially those with hydrops, should be referred to a fetal center for evaluation.

Purpose An artificial placenta (AP) utilizing extracorporeal life support (ECLS) could avoid the harm of mechanical ventilation (MV) while allowing the lungs to develop. Methods AP lambs (n = 5) were delivered at 118 days gestational age (GA; term = 145 days) and placed on venovenous ECLS (VV-ECLS) with jugular drainage and umbilical vein reinfusion. Lungs remained fluid-filled. After 10 days, lambs were ventilated. MV control lambs were delivered at 118 (“early MV”; n = 5) or 128 days (“late MV”; n = 5), and ventilated. Compliance and oxygenation index (OI) were calculated. After sacrifice, lungs were procured and HE alveolar development was quantified by the area fraction of alveolar septal tips staining double-positive for both markers. Results Compliance of AP lambs was 2.79 ± 0.81 Cdyn compared to 0.83 ± 0.19 and 3.04 ± 0.99 for early and late MV, respectively. OI in AP lambs was lower than early MV lambs (6.20 ± 2.10 vs. 36.8 ± 16.8) and lung injury lower as well (1.8 ± 1.6 vs. 6.0 ± 1.2). Double-positive area fractions were higher in AP lambs (0.012 ± 0.003) than early (0.003 ± 0.0005) and late (0.004 ± 0.002) MV controls. Conclusions Lung development continues and lungs are protected from injury during AP support relative to mechanical ventilation. Level of evidence n/a (basic/translational science).

Background Changes in the surgical training landscape have sparked an interest in developing new educational models anchored on entrustment assessment. We sought to optimize the validated OpTrust entrustment assessment tool by comparing ratings from short-course video reviews to previously validated intraoperative assessments. Methods Entrustment assessment scores for video-based and 1-h (short-course) observations were compared to previously validated intraoperative assessment scores. Faculty and residents were surveyed for their perceptions related to operative observation. Results There was a strong association between entrustment scores when comparing in-person to video-based observations (R2 = 0.76–0.84, p 97%) felt observation did not negatively impact operative experience. Conclusions Assessment of entrustment behaviors using short-course video review provides a feasible approach to intraoperative assessment. This latest application of OpTrust allows for the tool to be incorporated into surgical training programs across a variety of environments.

Purpose We evaluated whether brain development continues and brain injury is prevented during Artificial Placenta (AP) support utilizing extracorporeal life support (ECLS). Methods Lambs at EGA 118days (term=145; n=4) were placed on AP support (venovenous ECLS with jugular drainage and umbilical vein reinfusion) for 7days and sacrificed. Early (EGA 118; n=4) and late (EGA 127; n=4) mechanical ventilation (MV) lambs underwent conventional MV for up to 48h and were sacrificed, and early (n=5) and late (n=5) tissue control (TC) lambs were sacrificed at delivery. Brains were harvested, formalin-fixed, rehydrated, and studied by magnetic resonance imaging (MRI). The gyrification index (GI), a measure of cerebral folding complexity, was calculated for each brain. Diffusion-weighted imaging was used to determine fractional anisotropy (FA) and apparent diffusion coefficient (ADC) in multiple structures to assess white matter (WM) integrity. Results No intracranial hemorrhage was observed. GI was similar between AP and TC groups. ADC and FA did not differ between AP and late TC groups in any structure. Compared to late MV brains, AP brains demonstrated significantly higher ADC (0.45±0.08 vs. 0.27±0.11, p=0.02) and FA (0.61±0.04 vs. 0.44±0.05; p=0.006) in the cerebral peduncles. Conclusions After 7days of AP support, WM integrity is preserved relative to mechanical ventilation. Type of study Research study.

Purpose The Avalon dual-lumen venovenous catheter has several advantages, but placement techniques and management have not been adequately addressed in the pediatric population. We assessed our institutional outcomes and complications using the Avalon catheter in children. Methods We reviewed all pediatric patients who had Avalon catheters placed for respiratory failure at our institution, excluding congenital heart disease patients, from April 2009 to March 2016. All patients were managed using our standard ECMO protocol, and cannula position was followed by daily chest x-ray and intermittent echocardiography (ECHO). Data included demographics, diagnosis, PRISM3 predicted mortality, ECMO duration, complications, and survival. The primary outcome was the need for catheter repositioning. Results Twenty-five patients were included, with mean age 8.3 ± 6.9 years and 15 ± 22 days of ECMO support. Overall survival was 68% (17/25). Fourteen patients (56%) underwent placement with fluoroscopy in addition to ultrasound and ECHO, primarily after 2013. Overall, thirteen patients (52%) had problems with cannula malposition. 9 of these (69%) required cannula repositioning. Three of 14 (21%) cannulas placed with fluoroscopy required repositioning, compared to 7/11 (64%) placed without fluoroscopy (p = 0.05). Conclusions Complications are common with the Avalon catheter in children. Safe percutaneous access requires ultrasound guidance, and use of intraoperative fluoroscopy in addition to echocardiography decreases malposition rates. Level of evidence IV (Prognosis study).

Purpose Gastrostomy tubes (G-tubes) can be placed utilizing a variety of techniques. Here we present a case series to demonstrate feasibility of a novel method, ultrasound-guided G-tube placement (USGTP). Methods All cases of USGTP at our institution from September 2015–August 2016 were reviewed. Data included demographics, operative time, complications, time to first feeding, and 30-day readmissions. All steps of the procedure were carried out using ultrasound guidance, resulting in placement of a low-profile G-tube. Results Twelve patients underwent USGTP. Median age at operation was 2.6 years (IQR 0.9–5.3) and median weight 9.9 kg (IQR 7.2–18.4). Median operative time was 27 min. (IQR 20–44). First feeding occurred 8.8 ± 2.9 h after the procedure. The second patient in the series experienced the only operative complication. In this case, a linear probe was used with insufficient gastric distension, resulting in placement of the tube through a fold in the stomach wall. This was recognized and remedied intraoperatively. This prompted successful technique modification for future USGTPs. Only one patient was readmitted within 30 days, and this was related to urinary retention, an underlying problem. Conclusion US-guided G-tube placement appears initially to be safe, efficient and effective. Advantages include good anatomical delineation, a single incision, initial placement of a low-profile G-tube, and avoidance of endoscopy, laparoscopy, and radiation. This report illustrates feasibility of USGTP paving the way for further investigation and comparison to other existing gastrostomy insertion methods. Level of evidence IV.

Purpose Management guidelines for infants with chylothorax lack substantial evidence. We sought to identify variables that impact outcomes in these patients in order to develop an evidence-based management algorithm. Methods We retrospectively reviewed the medical records of all infants diagnosed with chylothorax from June 2005 to December 2014 at our institution. Data collected included demographics, chest tube output (CTO), medical and dietary interventions, surgical procedures, and absolute lymphocyte count (ALC). Outcomes analyzed included death, sepsis, necrotizing enterocolitis (NEC), requiring surgery, and success of therapy, defined as CTO decrease by >50% within 7days. Results Of 178 neonates with chylothorax, initial therapy was high medium chain triglyceride (MCT) feedings in 106 patients, nothing by mouth (NPO), total parenteral nutrition (TPN) in 21, and NPO/TPN plus octreotide in 45. Octreotide use in addition to NPO/TPN revealed no significant differences in any outcome including success (47% vs. 43%, p =0.77). Initial CTO and ALC correlated with needing surgery ( p =0.002 and p =0.006, respectively), and with death ( p =0.028 and p =0.043, respectively). ALC also correlated with sepsis ( p Conclusions Octreotide has no advantage over NPO/TPN alone in infants with chylothorax. CTO and ALC predict requiring surgery. We propose a management guideline based on CTO and ALC without a role for octreotide. Type of study Retrospective case–control study. Level of evidence 3

Fecal incontinence is a socially debilitating problem for many children. We hypothesized that in selected patients with medically-refractory encopresis, placement of an appendicostomy or cecostomy tube for administration of antegrade continence enemas (ACE) would improve quality of life (QOL).We reviewed all patients with encopresis who underwent appendicostomy or cecostomy placement from 2003 to 2014 at our institution. We contacted subjects' parents by phone and administered 3 surveys: a survey reflecting current stooling habits, a disease-specific QOL survey, and the PedsQL™ QOL survey. QOL surveys were completed twice by parents, once reflecting pre-operative QOL, then again reflecting current QOL. Pre-procedure and post-procedure scores were compared by paired t-test.Ten patients underwent appendicostomy/cecostomy for encopresis. Eight completed phone surveys. All procedures were performed laparoscopically. All patients experienced fecal soiling pre-operatively, whereas 5/8 surveyed patients (63%) noted complete resolution of soiling post-procedure (p0.01). General and disease-specific QOL improved from pre-procedure to post-procedure in the following domains: social habits, physical activity, ability to spend the night elsewhere, feeling, and overall QOL (p0.05). PedsQL™ scores improved significantly in physical functioning, social functioning, and overall functioning (p0.05).Antegrade continence enemas significantly improve quality of life in patients with medically-refractory encopresis, likely related to resolution of soiling.4.

Purpose Chronic obstructive defecation can occur in patients with Hirschsprung Disease (HD) and internal anal sphincter (IAS) achalasia. Injection of Botulinum Toxin (BoTox) into the IAS can temporarily relieve obstructive defecation, but can be challenging when performed by tactile sense alone. We compared results of BoTox injections with and without ultrasound (US) guidance. Methods We retrospectively reviewed BoTox injections into the IAS for obstructive defecation over 5years. Analyzed outcomes included short-term improvement, defined as resolution of enterocolitis, new ability to spontaneously defecate, and/or normalization of bowel movement frequency 2weeks post-operatively, as well as requirement of more definitive surgical therapy (myotomy/myomectomy, colectomy, colostomy, cecostomy/appendicostomy, and/or sacral nerve stimulator implantation). Outcomes were compared using t-test and Fisher's Exact test, with significance defined as p Results Twelve patients who underwent BoTox injection were included, including 5 patients who underwent injections both with and without ultrasound. Ten underwent an ultrasound-guided injection (13 injection procedures), 5 of whom had HD. Seven underwent an injection without ultrasound guidance (17 injection procedures), 5 of whom had HD. Procedures performed with US resulted in greater short-term improvement (76% versus 65% without ultrasound) and less requirement of a definitive procedure for obstructive defecation (p Conclusions US-guided BoTox injection is safe and effective for obstructive defecation, and may decrease the need for a definitive operation. Level of Evidence III.

Background Signaling lymphocytic activation molecule (SLAM)–associated protein (SAP) can mediate the function of SLAM molecules, which have been proposed to be involved in the development of autoimmunity in mice. Objective We sought to determine whether the SLAM/SAP pathway regulates the establishment of human B-cell tolerance and what mechanisms of B-cell tolerance could be affected by SAP deficiency. Methods We tested the reactivity of antibodies isolated from single B cells from SAP-deficient patients with X-linked lymphoproliferative disease (XLP). The expressions of SAP and SLAM family members were assessed in human bone marrow–developing B cells. We also analyzed regulatory T (Treg) cell function in patients with XLP and healthy control subjects. Results We found that new emigrant/transitional B cells from patients with XLP were enriched in autoreactive clones, revealing a defective central B-cell tolerance checkpoint in the absence of functional SAP. In agreement with a B cell–intrinsic regulation of central tolerance, we identified SAP expression in a discrete subset of bone marrow immature B cells. SAP colocalized with SLAMF6 only in association with clustered B-cell receptors likely recognizing self-antigens, suggesting that SLAM/SAP regulate B-cell receptor–mediated central tolerance. In addition, patients with XLP displayed defective peripheral B-cell tolerance, which is normally controlled by Treg cells. Treg cells in patients with XLP seem functional, but SAP-deficient T cells were resistant to Treg cell–mediated suppression. Indeed, SAP-deficient T cells were hyperresponsive to T-cell receptor stimulation, which resulted in increased secretion of IL-2, IFN-γ, and TNF-α. Conclusions SAP expression is required for the counterselection of developing autoreactive B cells and prevents their T cell–dependent accumulation in the periphery.

Objective To examine the frequency of allergic sensitization to staphylococcal superantigens in young children with mild to moderate atopic dermatitis (AD). Study design AD severity was assessed with objective Scoring AD. Serum IgE to staphylococcal enterotoxin (SE) A, SEB, SEC, SED, and toxic shock syndrome toxin-1 were measured with ImmunoCAP. Comparisons between mild AD and moderate AD were performed by using logistic regressions. Results The prevalence of allergic sensitization to staphylococcal superantigens in patients with mild and moderate AD was 38% and 63%, respectively. Allergic sensitization to staphylococcal superantigens, particularly SEA and SED, was found to be associated with moderate AD, compared with mild AD. Conclusions Our results suggest that allergic sensitization to staphylococcal superantigens is common even in young children with mild to moderate AD, and such sensitization may contribute to the disease severity of these patients.

HIV-infected children and youth will continue to be seen in emergency settings. Clinical recognition of patients possibly infected with HIV and the use of rapid HIV testing to identify them are of paramount importance. Effective management of patients known to be infected with HIV requires an understanding of appropriate infection control procedures, an appreciation of risk for opportunistic processes based upon patients' CD4+ T-cell counts, and access to consultation with expert HIV care providers. Finally, effectiveness of and indications for postexposure prophylaxis after occupational and nonoccupational exposures must be appreciated by emergency care providers.

Two HIV-1-infected children on antiretroviral therapy were enrolled into a clinical study of retroviral-mediated transfer of a gene that inhibits replication of HIV-1, targeting bone marrow CD34+ hematopoietic stem/progenitor cells. Two retroviral vectors were used, one encoding a "humanized" dominant-negative REV protein (huM10) that is a potent inhibitor of HIV-1 replication and one encoding a nontranslated marker gene (FX) to serve as an internal control for the level of gene marking. Peripheral blood mononuclear cells (PBMC) containing the huM10 gene or FX gene were detected by quantitative PCR at frequencies of approximately 1/10,000 in both subjects for the first 1-3 months following re-infusion of the gene-transduced bone marrow, but then were at or below the limits of detection (

Objective To produce a simple and effective instrument to evaluate and monitor the nutritional risk of children infected with the human immunodeficiency virus (HIV). Design The test instrument was developed in consultation with 5 physicians, 5 nutritionists, and 5 social workers with expertise in caring for HIV-infected children. Patient information was collected through medical record review for 19 sociodemographic, 10 anthropometric, 4 biochemical, 6 dietary intake, and 19 medical factors. As a part of routine nutrition care, anthropometric data were obtained and the caregiver was asked to complete a 3-day diet record. Also recorded were the most recent CD4+ T-cell numbers and serum HIV p24 antigen and plasma HIV RNA levels. Subjects/setting Thirty-nine HIV-infected children were selected using quota sampling; that is, subjects were stratified by clinical class as defined by the Centers for Disease Control and Prevention. Statistical analysis The severity or degree of potential nutritional risk in each section (anthropometric, biochemical, dietary intake, and medical data) was graded (0 to 4, 0=low risk) and summed. Reliability of internal consistency was determined through covariance matrixes. Validity was determined through Pearson product moment correlation coefficients to measure convergent and divergent validity; predictive validity was determined using analysis of variance. Correlation for validity was compared to 6 selected dependent variables: weight for height, weight growth velocity, lean body mass, serum albumin level, CD 4 + T-cell numbers, and quantitative plasma HIV RNA levels. Results Of the 38 factors that were analyzed for reliability, 11 fell in the strongly reliable range: height for age, weight for age, clinical class, somatic protein stores, mid-arm circumference, weight for height, serum albumin, immunologic status, body mass index, energy intake, and opportunistic infection. Conclusions Anthropometric, dietary intake, and medical data were reliable indicators of nutritional risk. The entire instrument was reliable after 8 of the weakest items were removed. The instrument was found to be valid and a good predictor of nutritional risk in HIV-infected children. J Am Diet Assoc. 2000; 100:323-329.

Standard serologic tests for human immunodeficiency (HIV)-specific antibodies are difficult to interpret in young infants born "at risk" for HIV infection owing to transplacentally acquired maternal IgG. This article reviews the treatment advantages of early diagnosis of HIV infection, the indications for HIV testing in children, the general principles and use of individual HIV assays, and an idealized evaluation schedule for infants born "at risk" for HIV infection.

Background Inhalation of pectin has been identified as a cause of occupational asthma. However, allergic reactions to orally ingested pectin have not been reported. Objectives To describe a child with pectin-induced food anaphylaxis and to discuss its possible relationship to cashew allergy. Methods A 3½-year-old boy developed anaphylaxis once after eating cashews and later after eating a pectin-containing fruit “smoothie.” He also has a history of generalized pruritus after eating grapefruit. Skin tests or radioallergosorbent tests (RASTs) were performed to pectin and other suspected food allergens. Results The child had a positive skin prick test reaction to pectin and a high RAST reaction to cashew and pistachio. He had a low-level positive RAST reaction to grapefruit. Results of allergy tests for the other potential food allergens were negative. The pectin in the smoothie was confirmed to be of citrus origin. Review of previous case reports of pectin-induced occupational asthma revealed several patients with allergies to and crossreactivity with cashew. Conclusions Ingestion, not only inhalation, of pectin can cause hypersensitivity reactions. Cashew, and possibly pistachio, allergy may be associated with pectin allergy, and the possibility of pectin allergy should be considered in cashew- or pistachio-allergic patients who have unexplained allergic reactions.

Introduction: Infliximab administered to women during pregnancy crosses the placenta and significant levels of these maternally-derived monoclonal antibodies remain detectable in infants for up to 6 months from birth. The effect of in-utero and perinatal infliximab exposure on immune development and response to vaccines is not known. The aim of this study was determine whether the offspring of mothers receiving infliximab during pregnancy have evidence of impaired immune function after birth as measured by specific antibody responses to standard immunizations. Methods: In this prospective study, blood collected at least 6 months following delivery from offspring with transplacental peripartum infliximab exposure were assessed for quantitative Immunoglobulin G, A, M; tetanus antibody titers and Haemophilus influenzae (Hib) type antibody titers. Maternal and infant infliximab levels and demographics were also collected. Results: The study cohort consisted of 8 infants born to women (7 Crohn's, 1 UC) receiving infliximab during pregnancy. The mean maternal age at the time of delivery was 32 years (range 22-38). The mean infusion interval during pregnancy was 5.6 weeks (4-8); 7 patients were on maintenance therapy and one received induction therapy. All received infliximab in the third trimester and 3 of the infants were breastfed. Mean gestational age was 39 weeks (range 36-41). All infants received a standard 6 month cycle of vaccines. Mean age of the infant at time of immune response assessment was 13 months (range 6 -28). Of 6 patients with available results, all had appropriate levels of IgG (mean 408 mg/dl) and IgA (mean 23.8 mg/dl). IgM levels (mean 47 mg/dl) were low (normal 34-126) in 4/6 patients studied. The range of Ig M levels for the infants with low levels were 17-29 mg/dl and they were 7-10 months of age. All 8 patients had an appropriate protective response to tetanus vaccine with a titer > 0.15. 7/8 patients had an appropriate response to Hib, while one 7 month old child failed to develop protective antibody levels (level = 0.36 [normal >1]). This latter child had a birth infliximab level of 8.7 mcg/ml and levels were undetectable by 3 months. Conclusions: Infants exposed to infliximab in-utero generally mount an appropriate response to routine childhood vaccinations. No child exhibited concerning immune deficits. The decreased level of IgM is of unclear significance.

The occurrence of two rare entities in a single patient can be fortuitous or may signify some deeper relationship. A young boy was recently treated for primary Candida meningitis. Autopsy findings suggested to an experienced pathologist the presence of chronic granulomatous disease (CGD), unrecognized during his life. The patient’s identical twin brother was tested and found to have the typical laboratory features of CGD. The literature on Candida meningitis was reviewed and 15 cases were discovered that apparently arose in the absence of recognized predisposing causes. All but one of these cases occurred in males, and most occurred during the first three decades of life. The case reports and literature review presented herein suggest that CGD should be suspected when a case of “primary” Candida meningitis is encountered.

Two preterm infant boys not known to be at risk developed clinical, laboratory, and pathologic features of acquired immune deficiency syndrome (AIDS) after receiving multiple blood transfusions in the neonatal period. Their clinical courses were characterized by failure to thrive, recurrent otitis media, hepatomegaly, and fatal interstitial pneumonia. Laboratory evaluation revealed progressive lymphopenia, reversed T helper/suppressor ratios, increased percentages of B-lymphocytes, decreased lymphoproliferative responses to mitogens, hyperimmunoglobulinemia, and high levels of circulating immune complexes. At postmortem examination thymic involution, lymphocyte depletion in spleen and lymph nodes, and micronodular mineralization in the central nervous system were seen. The findings were not specific for other known congenital immune deficiencies and were most indicative of AIDS. The lack of other risk factors suggests transmission of AIDS via blood transfusions in the neonatal period.

The critical role of the spleen in protecting subjects from systemic bacterial infection is well known. Plasma fibronectin (PF), cold-insoluble glubulin, and opsonic α 2 surface binding glycoprotein, has regulatory influence on reticuloendothelial system clearance activity and it is important for maximal phagocytosis. Ascorbic acid (AA) also appears to play an important role in phagocytic cell function. The purposes of this study were to determine the effects of spelenectomy and splenectomy with autoimplantation, with and without dietary AA supplementation, on PF levels. Six-week-old male Sprague-Dawley rats were divided into four experimental groups of 20 animals each: Nonoperated controls, sham-operated controls, totally splenectomized animals, and splenectomized animals with intraperitoneal autoimplantation. Each group was further randomly divided into two subgroups of 10 animals, those receiving dietary AA supplementation and those not receiving AA. PF levels were measured with a colorimetric assay immediately prior to and at 4 and 8 weeks after operation. Plasma AA determinations documented the effectiveness of dietary AA supplementation. PF levels in nonoperated and sham-operated controls increased significantly during the 8 weeks of experimentation. In contrast, PF levels decreased significantly following total splenectomy from 328±46 mcg/mL (mean±S.D.) to 285±46 at 4 weeks and rose to 303±77 at 8 weeks postoperatively in non-AA supplemented animals. Splenic autoimplantation eliminated this decrease in PF levels at 4 weeks. AA supplementation also protected against the decrease in PF levels in the splenectomized group. In the intragroup comparisons, AA supplementation did not produce a significant elevation of PF. Multiple immunologic defects have been noted after splenectomy. This study demonstrates an additional problem following splenectomy—a significant decrease in PF level.