Safety and Feasibility of Long-Distance Aeromedical Transport of Neonates and Children in Fixed-Wing Air Ambulance.

In cases of critical injury or illness abroad, fixed-wing air ambulance aircraft is employed to repatriate children to their home country. Air ambulance aircraft also transport children to foreign countries for treatment not locally available and newborns back home that have been born prematurely abroad. In this retrospective observational study, we investigated demographics, feasibility, and safety and outcomes of long-distance and international aeromedical transport of neonates and children. The study included 167 pediatric patients, 56 of those preterm neonates. A total of 41 patients were ventilated, 45 requiring oxygen prior to the transport, 57 transferred from an intensive care unit (ICU), and 48 to an ICU. Patients were transported by using Learjet 31A, Learjet 45, Learjet 55, and Bombardier Challenger 604, with a median transport distance of 1,008 nautical miles (NM), median transport time of 04:45 hours (median flight time = 03:00 hours), flight time ≥8 hours in 15 flights, and transport time ≥8 hours in 29 missions. All transports were accompanied by a pediatric physician/nurse team. An increase in FiO 2 during the transport was documented in 47/167 patients (28%). Therapy escalation (other than increased oxygen) was reported in 18 patients, and technical adverse events in 3 patients. No patient required CPR or died during the transport. Clinical transport outcome was rated by the accompanying physician as unchanged in 163 transports, improved in 4, and deteriorated in none. In summary, international, long-distance transport of neonatal and pediatric patients performed by experienced and well-equipped transport teams is feasible. Neither major adverse events nor physician-rated clinical deteriorations were observed in this group of patients.

Long-Distance Aeromedical Transport of Patients with COVID-19 in Fixed-Wing Air Ambulance Using a Portable Isolation Unit: Opportunities, Limitations and Mitigation Strategies.

INTRODUCTION: Aeromedical transport of patients with highly-infectious diseases, particularly over long distances with extended transport times, is a logistical, medical and organizational challenge. Following the 2014-2016 Ebola Crisis, sophisticated transport solutions have been developed, mostly utilizing large civilian and military airframes and the patient treated in a large isolation chamber. In the present COVID-19 pandemic, however, many services offer aeromedical transport of patients with highly-infectious diseases in much smaller portable medical isolation units (PMIU), with the medical team on the outside, delivering care through portholes. METHODS: We conducted a retrospective review of all transports of patients with proven or suspected COVID-19 disease, transported by Jetcall, Idstein, Germany, between April 1 and August 1, 2020, using a PMIU (EpiShuttle, EpiGuard AS, Oslo, Norway). Demographics and medical data were analyzed using the services' standardized transport protocols. Transport-associated challenges and optimization strategies were identified by interviewing and debriefing all transport teams after each transport. RESULTS: Thirteen patients with COVID-19 have been transported in a PMIU over distances up to 7,400 kilometers (km), with flight times ranging from 02:15 hours to 11:10 hours. We identified the main limitations of PMIU transports as limited access to the patient and reduced manual dexterity when delivering care through the porthole gloves and disconnection of lines and tubes during loading and unloading procedures. Technical solutions such as bluetooth-enabled stethoscopes, cordless ultrasound scanners and communication devices, meticulous preparation of the PMIU and the patient following standardized protocols and scenario-based training of crew members can reduce some of the risks. DISCUSSION: Transporting a patient with COVID-19 or any other highly infectious disease in a PMIU is a feasible option even over long distances, but adding a significant layer of additional risk, thus requiring a careful and individualized risk-benefit analysis for each patient prior to transport.

The role of magnetic resonance imaging in the diagnosis of central nervous system involvement in children with acute lymphoblastic leukemia.

PURPOSE: Acute lymphoblastic leukemia (ALL) is the most frequent malignancy in childhood. As central nervous system (CNS) involvement requires an intensified CNS-targeted therapy, timely diagnosis is essential. The aim of this retrospective analysis was to evaluate whether cranial magnetic resonance imaging (MRI) examinations findings correlate with cerebrospinal fluid (CSF) analysis on CNS involvement and whether MRI examinations reveal incidental findings with a clinical consequence. METHODS: All pediatric patients with ALL at our institution between 1998 and 2016 were identified. Patients were divided into two groups: de novo and relapsed ALL. Both groups were analyzed separately for the presence of CNS involvement. Incidental findings were also evaluated. RESULTS: Two hundred fifteen patients with de novo ALL and 31 with relapsed ALL were identified. In the de novo group, no patient was diagnosed CNS positive based on MRI results alone. In relapsed patients, only one patient had a positive MRI with negative CSF results and no neurological symptoms, thus was classified CNS positive solely on the basis of the MRI. In both groups, no patient showed an incidental finding that required therapy. CONCLUSION: In our study, MRI examinations do not improve the detection of CNS involvement compared with CSF analysis alone. In addition, the analysis of incidental findings does not add value to the performance of an MRI examination performed prior to treatment. Overall, MRI prior to treatment in pediatric patients with ALL is not necessary.

An Exponential Regression Model Reveals the Continuous Development of B Cell Subpopulations Used as Reference Values in Children.

B lymphocytes are key players in humoral immunity, expressing diverse surface immunoglobulin receptors directed against specific antigenic epitopes. The development and profile of distinct subpopulations have gained awareness in the setting of primary immunodeficiency disorders, primary or secondary autoimmunity and as therapeutic targets of specific antibodies in various diseases. The major B cell subpopulations in peripheral blood include naïve (CD19+ or CD20+IgD+CD27-), non-switched memory (CD19+ or CD20+IgD+CD27+) and switched memory B cells (CD19+ or CD20+IgD-CD27+). Furthermore, less common B cell subpopulations have also been described as having a role in the suppressive capacity of B cells to maintain self-tolerance. Data on reference values for B cell subpopulations are limited and only available for older age groups, neglecting the continuous process of human B cell development in children and adolescents. This study was designed to establish an exponential regression model to produce continuous reference values for main B cell subpopulations to reflect the dynamic maturation of the human immune system in healthy children.

Vemurafenib in Langerhans cell histiocytosis: report of a pediatric patient and review of the literature.

Selective BRAF inhibitors such as vemurafenib have become a treatment option in patients with Langerhans cell Histiocytosis (LCH). To date, only 14 patients receiving vemurafenib for LCH have been reported. Although vemurafenib can stabilize the clinical condition of these patients, it does not seem to cure the patients, and it is unknown, when and how to stop vemurafenib treatment. We present a girl with severe multisystem LCH who responded only to vemurafenib. After 8 months of treatment, vemurafenib was tapered and replaced by prednisone and vinblastine, a strategy which has not been described to date. Despite chemotherapy, early relapse occurred, but remission was achieved by re-institution of vemurafenib. Further investigation needs to address the optimal duration of vemurafenib therapy in LCH and whether and which chemotherapeutic regimen may prevent disease relapse after cessation of vemurafenib.

MECOM-associated syndrome: a heterogeneous inherited bone marrow failure syndrome with amegakaryocytic thrombocytopenia.

Heterozygous mutations in MECOM (MDS1 and EVI1 complex locus) have been reported to be causative of a rare association of congenital amegakaryocytic thrombocytopenia and radioulnar synostosis. Here we report on 12 patients with congenital hypomegakaryocytic thrombocytopenia caused by MECOM mutations (including 10 novel mutations). The mutations affected different functional domains of the EVI1 protein. The spectrum of phenotypes was much broader than initially reported for the first 3 patients; we found familial as well as sporadic cases, and the clinical spectrum ranged from isolated radioulnar synostosis with no or mild hematological involvement to severe bone marrow failure without obvious skeletal abnormality. The clinical picture included radioulnar synostosis, bone marrow failure, clinodactyly, cardiac and renal malformations, B-cell deficiency, and presenile hearing loss. No single clinical manifestation was detected in all patients affected by MECOM mutations. Radioulnar synostosis and B-cell deficiency were observed only in patients with mutations affecting a short region in the C-terminal zinc finger domain of EVI1. We propose the term MECOM-associated syndrome for this heterogeneous hereditary disease and inclusion of MECOM sequencing in the diagnostic workup of congenital bone marrow failure.

Crizotinib in ALK+ inflammatory myofibroblastic tumors-Current experience and future perspectives.

Inflammatory myofibroblastic tumor (IMT) and its subtype epithelioid inflammatory myofibroblastic sarcoma (EIMS) are rare soft-tissue tumors. As about 50% of IMT and 100% of EIMS contain activating rearrangements of the anaplastic lymphoma kinase (ALK) gene, targeted kinase inhibition of ALK by compounds such as crizotinib is a potential treatment option. We performed a literature review and analyzed a total of 30 patients with IMT/EIMS treated with crizotinib. A total of 12 patients achieved complete or partial remission. As preliminary data are promising, a prospective study evaluating crizotinib treatment in patients with unresectable/multifocal ALK+ IMT/EIMS is warranted.

Neurocognitive development in HIV-positive children is correlated with plasma viral loads in early childhood.

Because of neurocognitive impairments in perinatally human immunodeficiency virus (HIV)-infected children and adolescents, this study aimed to demonstrate the effect of plasma viral loads and early initiation of sufficient combined antiretroviral therapy (cART) on neurocognitive development.In total, 14 perinatally infected HIV-positive children (median age 8.24 years [range: 6.0-16.74]) receiving lopinavir/ritonavir (LPV/r)-based ART underwent neurocognitive testing using the Wechsler Intelligence Score for Children, 4th Edition (WISC-IV). All 14 patients participated in a pharmacokinetic study in which they were hospitalized for an entire day. As a child's ability to concentrate varies over the course of the day, all tests were performed in the morning.The patients' neurocognitive development did not significantly differ from the normative collective pattern for any of the following composite scores that were examined: full-scale intelligence quotient (IQ) (mean: 106.5, P = .1060), verbal comprehension index (mean: 106.0, P = .1356), perceptual reasoning index (mean: 106.0, P = .1357), working memory index (mean: 106.3, P = .1171), and processing speed index (mean: 98.1, P = .6313). The overall full-scale IQ scores were significantly higher in children who began ART within the first year of life (P = .0379), whereas low lopinavir/r plasma levels (P = .0070) and high viral load area under the curves (AUCs) in the first 3 years of life, but not later, significantly correlated with reduced neurocognitive performance (Spearman r = -0.64, P = .0278).In this cohort of cART treated HIV-positive children and adolescents, neurocognitive performance correlated with early and sufficient viral load suppression within the first 3 years of life.

Frequency and epitope specificity of anti-factor VIII C1 domain antibodies in acquired and congenital hemophilia A.

Several studies showed that neutralizing anti-factor VIII (anti-fVIII) antibodies (inhibitors) in patients with acquired hemophilia A (AHA) and congenital hemophilia A (HA) are primarily directed to the A2 and C2 domains. In this study, the frequency and epitope specificity of anti-C1 antibodies were analyzed in acquired and congenital hemophilia inhibitor patients (n = 178). The domain specificity of antibodies was studied by homolog-scanning mutagenesis (HSM) with single human domain human/porcine fVIII proteins and antibody binding to human A2, C1, and C2 domains presented as human serum albumin (HSA) fusion proteins. The analysis with HSA-fVIII domain proteins confirmed the results of the HSM approach but resulted in higher detection levels. The higher detection levels with HSA-fVIII domain proteins are a result of antibody cross-reactivity with human and porcine fVIII leading to false-negative HSM results. Overall, A2-, C1-, and C2-specific antibodies were detected in 23%, 78%, and 68% of patients with AHA (n = 115) and in 52%, 57%, and 81% of HA inhibitor patients (n = 63). Competitive binding of the human monoclonal antibody (mAb) LE2E9 revealed overlapping epitopes with murine C1-specific group A mAbs including 2A9. Mutational analyses identified distinct crucial binding residues for LE2E9 (E2066) and 2A9 (F2068) that are also recognized by anti-C1 antibodies present in patients with hemophilia. A strong contribution of LE2E9- and 2A9-like antibodies was particularly observed in patients with AHA. Overall, our study demonstrates that the C1 domain, in addition to the A2 and C2 domains, contributes significantly to the humoral anti-fVIII immune response in acquired and congenital hemophilia inhibitor patients.

Neutralisation of factor VIII inhibitors by anti-idiotypes isolated from phage-displayed libraries.

Following replacement therapy with coagulation factor VIII (FVIII), up to 30 % of haemophilia A patients develop FVIII-specific inhibitory antibodies (FVIII inhibitors). Immune tolerance induction (ITI) is not always successful, resulting in a need for alternative treatments for FVIII inhibitor-positive patients. As tolerance induction in the course of ITI appears to involve the formation of anti-idiotypes specific for anti-FVIII antibodies, such anti-idiotypes might be used to restore haemostasis in haemophilia A patients with FVIII inhibitors. We isolated anti-idiotypic antibody fragments (scFvs) binding to murine FVIII inhibitors 2-76 and 2-77 from phage-displayed libraries. FVIII inhibitor/anti-idiotype interactions were very specific as no cross-reactivity with other FVIII inhibitors or isotype controls was observed. ScFvs blocked binding of FVIII inhibitors to FVIII and neutralised their cognate inhibitors in vitro and a monoclonal mouse model. In addition, scFv JkH5 specific for FVIII inhibitor 2-76 stained 2-76-producing hybridoma cells. JkH5 residues R52 and Y226, located in complementary determining regions, were identified as crucial for the JkH5/2-76 interaction using JkH5 alanine mutants. SPR spectroscopy revealed that JkH5 interacts with FVIII inhibitor 2-76 with nanomolar affinity. Thus, FVIII inhibitor-specific, high-affinity anti-idiotypes can be isolated from phage-displayed libraries and neutralise their respective inhibitors. Furthermore, we show that anti-idiotypic scFvs might be utilised to specifically target inhibitor-specific B cells. Hence, a pool of anti-idiotypes could enable the reestablishment of haemostasis in the presence of FVIII inhibitors in patients or even allow the depletion of inhibitors by targeting inhibitor-specific B cell populations.

Analysis of T2 signal intensity helps in the differentiation between high and low-grade brain tumours in paediatric patients.

PURPOSE: Previous studies hypothesized that the analysis of magnetic resonance intensity of the solid portion in paediatric tumours can provide pre-surgical information about the histopathology. Classically, high signal-intensity in T2weighted (T2w) images identifies low-grade tumours, while anaplasia is characterized by T2 hypointensity. We aimed to investigate if T2w signal intensities can pre-operatively distinguish between low-grade and high-grade brain tumours in paediatric patients. METHODS: Two raters, blinded to the histological diagnosis, rated the signal intensity of MR images (T2w) from 36 children with newly diagnosed brain tumours, 17 children with low-grade brain tumours and 19 children with high-grade brain tumours were included in this study. Relative T2 values were obtained by dividing the T2w values of the solid portion of the tumour by the T2w values of the vitreous humour. RESULTS: The best cut-off point to distinguish low and high-grade paediatric brain tumours was 0.8. If the signal intensity was less than or equal to 0.8 the tumour was expected to be a high-grade tumour with a sensitivity of 100%. Prediction of a low-grade tumour was more uncertain with a sensitivity of 70.5%. Overall, 86% of the tumours would have been predicted correctly. CONCLUSION: Our data suggest that T2w signal intensities of the solid portion of brain tumours in paediatrics can pre-operatively differentiate between low-grade and high-grade tumours. In addition, T2 hypointensity may be helpful in targeting stereotactic biopsy.

Treatment of EBV-associated nodular sclerosing Hodgkin lymphoma in a patient with ataxia telangiectasia with brentuximab vedotin and reduced COPP plus rituximab.

Patients with ataxia telangiectasia (AT) with malignancies face poor prognosis due to increased treatment-related toxicity. Here, we report a 14-year-old male with AT and Hodgkin lymphoma (HL) who received brentuximab vedotin and reduced COPP plus rituximab courses. This treatment resulted in complete remission and showed no severe toxicity.

Epitope mapping via selection of anti-FVIII antibody-specific phage-presented peptide ligands that mimic the antibody binding sites.

The most serious complication in today's treatment of congenital haemophilia A is the development of neutralising antibodies (inhibitors) against factor VIII (FVIII). Although FVIII inhibitors can be eliminated by immune tolerance induction (ITI) based on repeated administration of high doses of FVIII, 20-30% of patients fail to become tolerant. Persistence of FVIII inhibitors is associated with increased morbidity and mortality. Data from recent studies provide evidence for a potential association between ITI outcome and epitope specificity of FVIII inhibitors. Nevertheless the determination of epitopes and their clinical relevance has not yet been established. In this study a general strategy for the identification of anti-FVIII antibody epitopes in haemophilia A patient plasma was to be demonstrated. Phage-displayed peptide libraries were screened against anti-FVIII antibodies to isolate specific peptides. Peptide specificity was confirmed by FVIII-sensitive ELISA binding. Peptide residues essential for antibody binding were identified by mutational analysis and epitopes were predicted via FVIII homology search. The proposed mapping strategy was validated for the monoclonal murine antibody (mAb) 2-76. Binding studies with FVIII variants confirmed the location of the predicted epitope at the level of individual amino acids. In addition, anti-FVIII antibody-specific peptide ligands were selected for 10 haemophilia A patients with FVIII inhibitors. Detailed epitope mapping for three of them showed binding sites on the A2, A3 and C2 domains. Precise epitope mapping of anti-FVIII antibodies using antibody-specific peptide ligands can be a useful approach to identify antigenic sites on FVIII.

Interleukin-2-stimulated natural killer cells are less susceptible to mycophenolate mofetil than non-activated NK cells: possible consequences for immunotherapy.

In a clinical phase I/II trial, pediatric patients with high-risk malignancies were treated with ex vivo IL-2-stimulated donor natural killer (NK) cells after transplantation with haploidentical stem cells. To evaluate the potential negative effects of the immunosuppressive drug mycophenolate mofetil (MMF) used for immunotherapy, the functionality and signaling of ex vivo NK cells was investigated. Our results show that during NK cell expansion, long-term (9 days) incubation with mycophenolic acid (MPA), the active metabolite of MMF, in therapeutically relevant concentrations led to the severe inhibition of NK cell proliferation. This correlated with a significantly reduced cytokine/chemokine secretion and the inhibited acquisition of surface receptors regarding cytotoxicity (e.g., NKp30, NKp44, NKp46, NKG2D), adhesion/migration (e.g., ICAM-1/CD54, LFA-1/CD11a, CD62L, CXCR3) and activation (e.g., CD25). Moreover, MPA prevented phosphorylation of the central signaling molecules STAT-3/-4/-5, AKT and ERK1/2. In contrast, short-term (24 h) MPA incubation of IL-2-stimulated NK cells had no or only marginal effects on the activated NK cell phenotype, including receptor expression, cytokine/chemokine secretion and intracellular signaling. Further, short-term MPA incubation only moderately affected the highly cytotoxic activity of previously IL-2-stimulated NK cells. In conclusion, while long-term MPA incubation significantly compromised ex vivo NK cell functionality, previously IL-2-activated NK cells seemed to be rather resistant to short-term MPA treatment. This finding supports the use of IL-2-activated NK cells as immunotherapy, especially for patients treated with MMF after haploidentical stem cell transplantation.

Eculizumab as first-line therapy for atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome (aHUS) is a genetic, life-threatening, chronic disease that can affect patients of all ages. aHUS is caused by uncontrolled complement activation due to genetic defects of complement regulation. Plasma exchange or infusion has been used to manage aHUS and may transiently maintain hematologic variables in some patients, but as the underlying complement dysregulation persists, end-stage renal disease or death occurs in 33% to 40% of patients during the first clinical manifestation. Here we present a pediatric case showing that first-line eculizumab treatment successfully blocked the progression of thrombotic microangiopathy in aHUS.

Conventional magnetic resonance imaging in the differentiation between high and low-grade brain tumours in paediatric patients.

OBJECTIVE: It has been described that hyperintensity in diffusion-weighted imaging (DWI) correlates with high-grade tumours, and high signal-intensity in T2-weighted (T2w) images identifies low-grade tumours. We aimed to investigate the potential of routine conventional MRI sequences, such as DWI and T2-w, to pre-operatively distinguish between low-grade and high-grade brain tumours in paediatric patients. MATERIAL AND METHODS: Two raters, blinded to the histological diagnosis, rated the aspect and signal intensity of MR images (T2w and DWI) from 37 children with newly diagnosed brain tumours. Histological diagnoses included 18 low-grade and 19 high-grade brain tumours. RESULTS: The inter-rater agreement was 81-95%. High-grade tumours were never hypointense on DWI and low-grade tumours were usually hyperintense on T2w. Specificity was 100% for low-grade tumours and 90% for high-grade tumours. About 95% of the high-grade tumours and about 70% of the low-grade tumours were correctly diagnosed. CONCLUSION: The combination of general morphological aspect of the tumours and signals on T2-w and DWI yield a high accuracy of pre-operative differentiation between low-grade and high-grade paediatric tumours.

Treatment of patients with advanced cancer with the natural killer cell line NK-92.

BACKGROUND AIMS: Natural killer (NK) cells, either naive or genetically engineered, are increasingly considered for cellular therapy of patients with malignancies. When using NK cells from peripheral blood, the number of expanded NK cells can be highly variable and the need for NK cell enrichment can make the process expensive. The NK-92 cell line (CD56+/CD3-) that was isolated from a patient with lymphoma has predictable high cytotoxic activity and can be expanded under good manufacturing practice conditions in recombinant interleukin-2. METHODS: Fifteen patients (age, 9-71 years) with advanced, treatment-resistant malignancies, either solid tumors/sarcomas (n = 13) or leukemia/lymphoma (n = 2), received two infusions of NK-92 cells, given 48 h apart. Three cohorts of patients were treated with escalating doses of NK-92 cells (n = 7 at 1 × 10(9), n = 6 at 3 × 10(9) and n = 2 at 1 × 10(10) cells/m(2)). RESULTS: No infusion-related or long-term side effects were observed. The dose of 10(10) cells/m(2) was considered the maximum expandable cell dose with the use of an established culture bag system. Three fourths of patients with lung cancer had some anti-tumor response. Only one patient of seven had development of human leukocyte antigen antibodies. The persistence of NK-92 cells (male origin) in the circulation was confirmed by Y chromosome-specific polymerase chain reaction in two female patients. CONCLUSIONS: Infusions of NK-92 cells up to 10(10) cells/m(2) were well tolerated. Despite the allogeneic nature of NK-92, development of human leukocyte antigen antibodies in these patients with cancer appears to be rare. The cells can persist in the recipient's circulation for at least 48 h. Some encouraging responses were seen in patients with advanced lung cancer.

Clinical grade purification and expansion of NK cell products for an optimized manufacturing protocol.

Allogeneic natural killer (NK) cells are used for adoptive immunotherapy after stem cell transplantation. In order to overcome technical limitations in NK cell purification and activation, the following study investigates the impact of different variables on NK cell recovery, cytotoxicity, and T-cell depletion during good manufacturing practice (GMP)-grade NK cell selection. Forty NK cell products were derived from 54 unstimulated donor leukaphereses using immunomagnetic CD3 T-cell depletion, followed by a CD56 cell enrichment step. For T-cell depletion, either the depletion 2.1 program in single or double procedure (D2.11depl, n = 18; D2.12depl, n = 13) or the faster depletion 3.1 (D3.1, n = 9) was used on the CliniMACS instrument. Seventeen purified NK cell products were activated in vitro by IL-2 for 12 days. The whole process resulted in a median number of 7.59 × 10(8) CD56(+)CD3(-) cells with both purity and viability of 94%, respectively. The T-cell depletion was significantly better using D2.11depl/2depl compared to D3.1 (log 4.6/log 4.9 vs. log 3.7; p < 0.01) and double procedure in two stages led always to residual T cells below 0.1%. In contrast D3.1 was superior to D2.11depl/2depl with regard to recovery of CD56(+)CD3(-) NK cells (68% vs. 41%/38%). Concomitant monocytes and especially IL-2 activation led to increased NK cell activity against malignant target cells compared to unstimulated NK cells, which correlated with both up-regulation of natural cytotoxicity receptors and intracellular signaling. Overall, wide variations in the NK cell expansion rate and the distribution of NK cell subpopulations were found. In conclusion, our results indicate that GMP-grade purification of NK cells might be improved by a sequential processing of T-cell depletion program D2.1 and D3.1. In addition NK cell expansion protocols need to be further optimized.