Publications by authors named "Rupert Handgretinger"

309 Publications

Immunomonitoring of Stage IV Relapsed Neuroblastoma Patients Undergoing Haploidentical Hematopoietic Stem Cell Transplantation and Subsequent GD2 (ch14.18/CHO) Antibody Treatment.

Front Immunol 2021 22;12:690467. Epub 2021 Jul 22.

Department of Pediatric Hematology and Oncology, University Children's Hospital, Eberhard Karls University Tuebingen, Tuebingen, Germany.

Haploidentical stem cell transplantation (haplo SCT) in Stage IV neuroblastoma relapsed patients has been proven efficacious, while immunotherapy utilizing the anti-GD2 antibody dinutuximab beta has become a standard treatment for neuroblastoma. The combinatorial therapy of haplo SCT and dinutuximab may potentiate the efficacy of the immunotherapy. To gain further understanding of the synergistic effects, functional immunomonitoring was assessed during the clinical trial CH14.18 1021 Antibody and IL2 After haplo SCT in Children with Relapsed Neuroblastoma (NCT02258815). Rapid immune reconstitution of the lymphoid compartment was confirmed, with clinically relevant dinutuximab serum levels found in all patients over the course of treatment. Only one patient developed human anti-chimeric antibodies (HACAs). In-patient monitoring revealed highly functional NK cell posttransplant capable of antibody-dependent cellular cytotoxicity (ADCC). Degranulation of NK cell subsets revealed a significant response increased by dinutuximab. This was irrespective of the KIR receptor-ligand constellation within the NK subsets, defined by the major KIR receptors CD158a, CD158b, and CD158e. Moreover, complement-dependent cytotoxicity (CDC) was shown to be an extremely potent effector-cell independent mechanism of tumor cell lysis, with a clear positive correlation to GD2 expression on the cancer cells as well as to the dinutuximab concentrations. The testing of patient-derived effector cells and the sera collected during dinutuximab therapy demonstrated both high functionality of the newly established lymphoid immune compartment and provided confidence that the antibody dosing regimen was sufficient over the duration of the dinutuximab therapy (up to nine cycles in a 9-month period). During the course of the dinutuximab therapy, proinflammatory cytokines and markers (sIL2R, TNFa, IL6, and C reactive protein) were significantly elevated indicating a strong anti-GD2 immune response. No impact of FcGR polymorphism on event-free and overall survival was found. Collectively, this study has shown that in-patient functional immunomonitoring is feasible and valuable in contributing to the understanding of anti-cancer combinatorial treatments such as haplo SCT and antibody immunotherapy.
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http://dx.doi.org/10.3389/fimmu.2021.690467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8339919PMC
July 2021

Myeloid-Derived Suppressor Cells Dampen Airway Inflammation Through Prostaglandin E2 Receptor 4.

Front Immunol 2021 12;12:695933. Epub 2021 Jul 12.

Department of Experimental and Clinical Pharmacology and Pharmacogenomics, University Hospital Tübingen, Tübingen, Germany.

Emerging evidence suggests a mechanistic role for myeloid-derived suppressor cells (MDSCs) in lung diseases like asthma. Previously, we showed that adoptive transfer of MDSCs dampens lung inflammation in murine models of asthma through cyclooxygenase-2 and arginase-1 pathways. Here, we further dissected this mechanism by studying the role and therapeutic relevance of the downstream mediator prostaglandin E2 receptor 4 (EP4) in a murine model of asthma. We adoptively transferred MDSCs generated using an EP4 agonist in a murine model of asthma and studied the consequences on airway inflammation. Furthermore, pegylated human arginase-1 was used to model MDSC effector activities. We demonstrate that the selective EP4 agonist L-902,688 increased the number and suppressive activity of MDSCs through arginase-1 and nitric oxide synthase-2. These results showed that adoptive transfer of EP4-primed MDSCs, EP4 agonism alone or arginase-1 administration ameliorated lung inflammatory responses and histopathological changes in asthmatic mice. Collectively, our results provide evidence that MDSCs dampen airway inflammation in murine asthma through a mechanism involving EP4.
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http://dx.doi.org/10.3389/fimmu.2021.695933DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8311661PMC
July 2021

Hematopoietic Stem Cell Transplantation for Patients with Autosomal Recessive Complete INF-λ Receptor 2 Deficiency: Experience in Oman.

Transplant Cell Ther 2021 Jul 20. Epub 2021 Jul 20.

Department of Hematology, Sultan Qaboos University Hospital, Muscat, Oman; Hematology Section, Apollo Hospital Muscat, Oman. Electronic address:

Autosomal recessive complete INF-γ receptor-2 (IFN-γR2) deficiency is a rare, potentially fatal primary immune deficiency that predisposes to disseminated mycobacterial disease. Hematopoietic stem cell transplantation (HSCT) is currently the only curative treatment. Few patients have been reported so far. Here we report the outcomes of HSCT in 7 patients with IFNγ-R2 deficiency from 3 Omani families who underwent HSCT at Sultan Qaboos University Hospital in Oman. All patients were homozygous for the same mutation (c.-175_+102del) of INFGR2. Four patients underwent HLA-matched related donor (MRD) HSCT (3 siblings and 1 parent), and the other 3 underwent T cell-depleted (TCD) haploidentical HSCT from a family donor. The stem cell source was peripheral blood stem cells in 5 patients and bone marrow in 2 patients. Five patients received myeloablative conditioning, and 2 had reduced-intensity conditioning. The overall survival rate was 85.7%, and the event-free survival was 71.4%. One of the 7 patients died on day +31 with gram-negative sepsis, and the other 6 patients were cured from their original disease (median follow-up of 78.5 months). One patient had primary graft failure following a TCD-haploidentical transplantation and underwent successful retransplantation from another haploidentical relative. Three patients received a donor lymphocyte infusion for mixed chimerism. Our findings indicate that HSCT is curative for complete IFN-γR2 deficiency. In this cohort from Oman, 85.7% of the patients were cured with either an MRD or a TCD haploidentical transplantation. Genetic analysis at birth in children of high-risk couples permits early diagnosis, prevents the morbidity of BCG vaccination, and can enable safer and more successful transplantation outcomes.
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http://dx.doi.org/10.1016/j.jtct.2021.07.013DOI Listing
July 2021

Protective T cell receptor identification for orthotopic reprogramming of immunity in refractory virus infections.

Mol Ther 2021 May 29. Epub 2021 May 29.

Department of Pediatric Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Dr. von Hauner Children's Hospital, University Hospital, LMU Munich, Munich Germany; Department I - General Pediatrics, Hematology/Oncology, University Hospital Tubingen, Children's Hospital, Tubingen, Germany; German Center for Infection Research (DZIF), Partner Site Munich, Munich, Germany. Electronic address:

Viral infections cause life-threatening disease in immunocompromised patients and especially following transplantation. T cell receptor (TCR) engineering redirects specificity and can bring significant progress to emerging adoptive T cell transfer (ACT) approaches. T cell epitopes are well described, although knowledge is limited on which TCRs mediate protective immunity. In this study, refractory adenovirus (AdV) infection after hematopoietic stem cell transplantation (HSCT) was treated with ACT of highly purified Hexon5-specific T cells using peptide major histocompatibility complex (pMHC)-Streptamers against the immunodominant human leukocyte antigen (HLA)-A∗0101-restricted peptide LTDLGQNLLY. AdV was successfully controlled through this oligoclonal ACT. Novel protective TCRs were isolated ex vivo and preclinically engineered into the TCR locus of allogeneic third-party primary T cells by CRISPR-Cas9-mediated orthotopic TCR replacement. Both TCR knockout and targeted integration of the new TCR in one single engineering step led to physiological expression of the transgenic TCR. Reprogrammed TCR-edited T cells showed strong virus-specific functionality such as cytokine release, effector marker upregulation, and proliferation capacity, as well as cytotoxicity against LTDLGQNLLY-presenting and AdV-infected targets. In conclusion, ex vivo isolated TCRs with clinical proven protection through ACT could be redirected into T cells from naive third-party donors. This approach ensures that transgenic TCRs are protective with potential off-the-shelf use and widened applicability of ACT to various refractory emerging viral infections.
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http://dx.doi.org/10.1016/j.ymthe.2021.05.021DOI Listing
May 2021

Iodine-GD2-ch14.18 scintigraphy to evaluate option for radioimmunotherapy in patients with advanced tumors.

J Nucl Med 2021 May 28. Epub 2021 May 28.

Department of Nuclear Medicine and Clinical Molecular Imaging, University Hospital Tuebingen, Germany.

The tumor-selective ganglioside antigene GD2 is frequently expressed on neuroblastomas and to a lesser extent also on sarcomas and neuroendocrine tumors. Aim of our study was to evaluate tumor targeting and biodistribution of iodine-131-labeled chimeric GD2-antibody clone 14/18 (I-GD2-ch14.18) in patients with late-stage disease in order to identify eligibility for radioimmunotherapy. 20 patients (neuroblastoma = 9; sarcoma = 9; pheochromocytoma = 1, neuroendocrine tumor = 1) were involved in this study. 21 to 131 MBq (1-2 MBq/kg) of I-131-GD2-ch14.18 (0.5 -1.0 mg) were injected intravenously. Planar scintigraphy was performed within 1 h from injection (d0), on d1, d2, d3, and d6 or d7 to analyse tumor uptake and tracer biodistribution. Serial blood samples were collected in 4 individuals. Irradiation dose to tumor lesions and organs was calculated using Olinda® software. The tumor targeting rate on a per-patient base was 65% (13/20) with 6/9 neuroblastomas showing uptake of I-GD2-ch14.18. Tumor lesions showed maximum uptake at 20-64 h p.i. (effective half-life in tumors 33-192 h). The tumor irradiation dose varied between 0.52 and 30.2 mGy/MBq (median: 9.08, = 13). Visual analysis showed prominent blood pool activity up to d2/d3 p.i.. No pronounced uptake was observed in the bone marrow compartment or in the kidneys. Bone marrow dose was calculated at 0.07-0.47 mGy/MBq (median: 0.14) while blood dose was 1.1-4.7 mGy/MBq. Two patients (1 neuroblastoma and 1 pheochromocytoma) with particularly high tumor uptake underwent radioimmunotherapy using 2.3 and 2.9 GBq of I-GD2-ch14.18 both achieving stable disease. Overall survival was 17 and 6 months, respectively. I-GD2-ch14.18 is cleared slowly from blood resulting in good tumor to background contrast not until 2 d after application. With acceptable red marrow and organ dose, radioimmunotherapy is an option for patients with high tumor uptake. However, due to the variable GD2-expression, decision should be made depending on pretherapeutic dosimetry.
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http://dx.doi.org/10.2967/jnumed.120.261854DOI Listing
May 2021

Removal of CD276 cells from haploidentical memory T-cell grafts significantly lowers the risk of GVHD.

Bone Marrow Transplant 2021 May 11. Epub 2021 May 11.

Department of Hematology and Oncology, Children's Hospital, Universität Tübingen, Tübingen, Germany.

Detrimental graft-versus-host disease (GVHD) still remains a major cause of death in hematopoietic stem cell transplantation (HSCT). The recently explored depletion of naive cells from mobilized grafts (CD45RA depletion) has shown considerable promise, yet is unable to eliminate the incidence of GVHD. Analysis of CD45RA-depleted haploidentical mixed lymphocytes culture (haplo-MLC) revealed insufficient suppression of alloresponses in the CD4 compartment and identified CD276 as a marker for alloreactive memory Th1 T cells. Conclusively, depleting CD276 cells from CD45RA-depleted haplo-MLC significantly attenuated alloreactivity to recipient cells while increasing antiviral reactivity and maintaining anti-third party reactivity in vitro. To evaluate these findings in vivo, bulk, CD45RA-depleted, or CD45RA/CD276-depleted CD4 T cells from HLA-DR4 healthy humans were transplanted into NSG-Ab°DR4 mice, a sensitive human allo-GVHD model. Compellingly, CD45RA/CD276-depleted grafts from HLA-DR4 donors or in vivo depletion of CD276 cells after transplant of HLA-DR4 memory CD4 T cells significantly delay the onset of GVHD symptoms and significantly alleviate its severity in NSG-Ab°DR4 mice. The clinical courses correlated with diminished Th1-cytokine secretion and downregulated CXCR6 expression of engrafted peripheral T cells. Collectively, mismatched HLA-mediated GVHD can be controlled by depleting recipient-specific CD276 alloreacting T cells from the graft, highlighting its application in haplo-HSCT.
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http://dx.doi.org/10.1038/s41409-021-01307-9DOI Listing
May 2021

In Vitro Evaluation of CD276-CAR NK-92 Functionality, Migration and Invasion Potential in the Presence of Immune Inhibitory Factors of the Tumor Microenvironment.

Cells 2021 04 26;10(5). Epub 2021 Apr 26.

Department of Hematology and Oncology, Children's Hospital, University Hospital Tuebingen, 72076 Tuebingen, Germany.

Background: Melanoma is the most lethal of all skin-related cancers with incidences continuously rising. Novel therapeutic approaches are urgently needed, especially for the treatment of metastasizing or therapy-resistant melanoma. CAR-modified immune cells have shown excellent results in treating hematological malignancies and might represent a new treatment strategy for refractory melanoma. However, solid tumors pose some obstacles for cellular immunotherapy, including the identification of tumor-specific target antigens, insufficient homing and infiltration of immune cells as well as immune cell dysfunction in the immunosuppressive tumor microenvironment (TME).

Methods: In order to investigate whether CAR NK cell-based immunotherapy can overcome the obstacles posed by the TME in melanoma, we generated CAR NK-92 cells targeting CD276 (B7-H3) which is abundantly expressed in solid tumors, including melanoma, and tested their effectivity in vitro in the presence of low pH, hypoxia and other known factors of the TME influencing anti-tumor responses. Moreover, the CRISPR/Cas9-induced disruption of the inhibitory receptor NKG2A was assessed for its potential enhancement of NK-92-mediated anti-tumor activity.

Results: CD276-CAR NK-92 cells induced specific cytolysis of melanoma cell lines while being able to overcome a variety of the immunosuppressive effects normally exerted by the TME. NKG2A knock-out did not further improve CAR NK-92 cell-mediated cytotoxicity.

Conclusions: The strong cytotoxic effect of a CD276-specific CAR in combination with an "off-the-shelf" NK-92 cell line not being impaired by some of the most prominent negative factors of the TME make CD276-CAR NK-92 cells a promising cellular product for the treatment of melanoma and beyond.
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http://dx.doi.org/10.3390/cells10051020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8145105PMC
April 2021

Universal Gene Correction Approaches for β-hemoglobinopathies Using CRISPR-Cas9 and Adeno-Associated Virus Serotype 6 Donor Templates.

CRISPR J 2021 04;4(2):207-222

University Children's Hospital, Department of Pediatrics I, Hematology and Oncology, University of Tübingen, Germany; University of Tübingen, Tübingen, Germany.

Mutations in the human gene are the cause of β-hemoglobinopathies, one of the most common inherited single-gene blood disorders in the world. Novel therapeutic approaches are based on lentiviral vectors (LVs) or CRISPR-Cas9-mediated gene disruption to express adult hemoglobin (HbA), or to reactivate the completely functional fetal hemoglobin, respectively. Nonetheless, LVs present a risk of insertional mutagenesis, while gene-disrupting transcription factors (BCL11A, KLF1) involved in the fetal-to-adult hemoglobin switch might generate dysregulation of other cellular processes. Therefore, universal gene addition/correction approaches combining CRISPR-Cas9 and homology directed repair (HDR) by delivering a DNA repair template through adeno-associated virus could mitigate the limitations of both lentiviral gene transfer and gene disruption strategies, ensuring targeted integration and controlled transgene expression. In this study, we attained high rates of gene addition (up to 12%) and gene correction (up to 38%) in hematopoietic stem and progenitor cells from healthy donors without any cell sorting/enrichment or the application of HDR enhancers. Furthermore, these approaches were tested in heterozygous (β/β) and homozygous (β/β, β/β) β-thalassemia patients, achieving a significant increase in HbA and demonstrating the universal therapeutic potential of this study for the treatment of β-hemoglobinopathies.
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http://dx.doi.org/10.1089/crispr.2020.0141DOI Listing
April 2021

RNA ImmunoGenic Assay: A Method to Detect Immunogenicity of Transcribed mRNA in Human Whole Blood.

Bio Protoc 2020 Dec 20;10(24):e3850. Epub 2020 Dec 20.

Department of Pediatrics I, Pediatric Infectiology and Immunology, Translational Genomics and Gene Therapy in Pediatrics, University of Tuebingen, Germany.

The mRNA therapeutics is a new class of medicine to treat many various diseases. However, transcribed (IVT) mRNA triggers immune responses due to recognition by human endosomal and cytoplasmic RNA sensors, but incorporation of modified nucleosides have been shown to reduce such responses. Therefore, an assay signifying important aspects of the human immune system is still required. Here, we present a simple method called 'RNA ImmunoGenic Assay' to measure immunogenicity of IVT-mRNAs in human whole blood. Chemically modified and unmodified mRNA are complexed with a transfection reagent (TransIT), and co-incubated in human whole blood. Specific cytokines are measured (TNF-α, INF-α, INF-γ, IL-6 and IL-12p70) using ELISAs. The qPCR analysis is performed to reveal the activation of specific immune pathways. The RNA ImmunoGenic Assay provides a simple and fast method to detect donor specific - immune response against mRNA therapeutics. .
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http://dx.doi.org/10.21769/BioProtoc.3850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8032499PMC
December 2020

Adapter Chimeric Antigen Receptor (AdCAR)-Engineered NK-92 Cells for the Multiplex Targeting of Bone Metastases.

Cancers (Basel) 2021 Mar 5;13(5). Epub 2021 Mar 5.

Department of Hematology and Oncology, University Hospital Tuebingen, Children's Hospital, 72076 Tuebingen, Germany.

Background: Since metastatic spreading of solid tumor cells often leads to a fatal outcome for most cancer patients, new approaches for patient-individualized, targeted immunotherapy are urgently needed.

Methods: Here, we established cell lines from four bone metastases of different tumor entities. We assessed AdCAR NK-92-mediated cytotoxicity in vitro in standard cytotoxicity assays as well as 3D spheroid models Results: AdCAR-engineered NK-92 cells successfully demonstrated distinct and specific cytotoxic potential targeting different tumor antigens expressed on cell lines established from bone metastases of mammary, renal cell and colorectal carcinoma as well as melanomas. In that process AdCAR NK-92 cells produced a multitude of NK effector molecules as well as pro inflammatory cytokines. Furthermore, AdCAR NK-92 showed increased cytotoxicity in 3D spheroid models which can recapitulate in vivo architecture, thereby bridging the gap between in vitro and in vivo models.

Conclusions: AdCAR NK-92 cells may provide an interesting and promising "off-the-shelf" cellular product for the targeted therapy of cancers metastasizing to the bone, while utilization of clinically approved, therapeutic antibodies, as exchangeable adapter molecules can facilitate quick clinical translation.
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http://dx.doi.org/10.3390/cancers13051124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7961358PMC
March 2021

Mesenchymal Stem Cell Therapy for Severe COVID-19 ARDS.

J Intensive Care Med 2021 Jun 5;36(6):681-688. Epub 2021 Mar 5.

Department of Anaesthesiology and Intensive Care Medicine, University Hospital Tübingen, 9188Eberhard-Karls-University, Tübingen, Germany.

Background: The COVID-19 pandemic reached Germany in spring 2020. No proven treatment for SARS-CoV-2 was available at that time, especially for severe COVID-19-induced ARDS. We determined whether the infusion of mesenchymal stromal cells (MSCs) would help to improve pulmonary function and overall outcome in patients with severe COVID-19 ARDS. We offered MSC infusion as an extended indication to all critically ill COVID-19 patients with a Horovitz index <100. We treated 5 out of 23 patients with severe COVID-19 ARDS with an infusion of MSCs. One million MSCs/kg body weight was infused over 30 minutes, and the process was repeated in 3 patients twice and in 2 patients 3 times.

Result: Four out of 5 MSC-treated patients compared to 50% of control patients (9 out of 18) received ECMO support (80%). The MSC group showed a higher Murray score on admission than control patients, reflecting more severe pulmonary compromise (3.5 ± 0.2 versus 2.8 ± 0.3). MSC infusion was safe and well tolerated. The MSC group had a significantly higher Horovitz score on discharge than the control group. Compared to controls, patients with MSC treatment showed a significantly lower Murray score upon discharge than controls. In the MSC group, 4 out of 5 patients (80%) survived to discharge and exhibited good pulmonary function, whereas only 8 out of 18 patients (45%) in the control group survived to discharge.

Conclusion: MSC infusion is a safe treatment for COVID-19 ARDS that improves pulmonary function and overall outcome in this patient population.
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http://dx.doi.org/10.1177/0885066621997365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8145440PMC
June 2021

Arsenic trioxide in pediatric cancer - a case series and review of literature.

Pediatr Hematol Oncol 2021 Aug 26;38(5):471-485. Epub 2021 Feb 26.

Pediatric Hematology/Oncology, Department of Pediatrics, University Hospital Tuebingen, Tuebingen, Germany.

Arsenic trioxide (ATO) has become an established component of treatment protocols for acute promyelocytic leukemia (APL) with excellent efficacy and no relevant sustained toxicity. Part of its action has been attributed to the inhibition of Hedgehog signaling (Hh) which enables a possible therapeutic approach as many pediatric tumor entities have been associated with increased Hh activity. We retrospectively analyzed 31 patients with refractory and relapsed pediatric cancer who were treated with ATO at the University Children's Hospital of Tuebingen. Additionally a literature review on the clinical and preclinical use of ATO in pediatric cancer treatment was performed.ATO alone as well as combinations with other drugs have proven effective and in mouse models of various pediatric malignancies. However, only few data on the clinical use of ATO in pediatric patients besides APL exist. In our patient sample, ATO was overall well tolerated in the treatment of various pediatric cancers, even in combination with other cytostatic drugs. Due to distinct tumor entities, differently progressed disease stages and varying co-medication, no clear statement can be made regarding the efficacy of ATO treatment. However, patients with proven Hh activation in molecular tumor profiling surpassed all other patients, who received ATO in an experimental treatment setting, in terms of survival. As molecular profiling of tumors increases and enhanced Hh activity can be detected at an early stage, ATO might expand its clinical use to other pediatric malignancies beyond APL depending on further clinical studies.
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http://dx.doi.org/10.1080/08880018.2021.1872748DOI Listing
August 2021

Severe SARS-CoV-2 Infection Inhibits Fibrinolysis Leading to Changes in Viscoelastic Properties of Blood Clot: A Descriptive Study of Fibrinolysis in COVID-19.

Thromb Haemost 2021 Feb 25. Epub 2021 Feb 25.

Center for Clinical Transfusion Medicine, University Hospital of Tübingen, Tübingen, Germany.

Background:  Accumulating evidence indicates toward an association between severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and procoagulatory state in blood. Thromboelastographic investigations are useful point-of-care devices to assess coagulation and fibrinolysis.

Objectives:  We investigated the hypothesis that the procoagulatory state in COVID-19 patients is associated with impaired fibrinolysis system.

Methods:  Altogether, 29 COVID-19 patients admitted to normal wards or to the intensive care unit (ICU) were included in this descriptive study. Whole blood samples were investigated by thromboelastography to assess coagulation and fibrinolysis. Additionally, standard routine coagulation testing and immunoassays for factors of fibrinolysis as plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA), plasminogen activity and α2-antiplasmin (A2AP) were performed.

Results:  A significantly increased lysis resistance and a significantly longer time of lysis after adding tissue plasminogen activator were observed in blood samples from ICU COVID-19 patients compared with healthy controls (maximal lysis: 3.25 ± 0.56 vs. 6.20 ± 0.89%,  = 0.0127; lysis time: 365.7 ± 44.6 vs. 193.2 ± 16.3 seconds,  = 0.0014). PAI-1 activity was significantly higher in plasma samples of ICU COVID-19 patients (PAI-1: 4.92 ± 0.91 vs. 1.28 ± 0.33 U/mL,  = 0.001). A positive correlation between the activity of PAI-1 and lysis time of the formed clot ( = 0.70,  = 0.0006) was observed.

Conclusion:  Our data suggest that severe SARS-CoV-2 infection is associated with impaired fibrinolytic activity in blood, where fibrinolytic inhibitors are elevated leading to an increased resistance to clot lysis. Thromboelastography could offer a tool to investigate the contribution of the fibrinolytic status to the procoagulatory condition in COVID-19.
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http://dx.doi.org/10.1055/a-1400-6034DOI Listing
February 2021

Allogeneic hematopoietic stem cell transplantation in leukocyte adhesion deficiency type I and III.

Blood Adv 2021 01;5(1):262-273

Pediatric Hematology, Ankara Child Health and Diseases Hematology Oncology Training and Research Hospital, Ankara, Turkey.

Type I and III leukocyte adhesion deficiencies (LADs) are primary immunodeficiency disorders resulting in early death due to infections and additional bleeding tendency in LAD-III. The curative treatment of LAD-I and LAD-III is allogeneic hematopoietic stem cell transplantation (allo-HSCT). In this retrospective multicenter study, data were collected using the European Society for Blood and Marrow Transplantation registry; we analyzed data from 84 LAD patients from 33 centers, all receiving an allo-HSCT from 2007 to 2017. The 3-year overall survival estimate (95% confidence interval [CI]) was 83% (74-92) for the entire cohort: 84% (75-94) and 75% (50-100) for LAD-I and LAD-III, respectively. We observed cumulative incidences (95% CI) of graft failure (GF) at 3 years of 17% (9%-26%) and grade II to IV acute graft-versus-host disease (aGVHD) at 100 days of 24% (15%-34%). The estimate (95% CI) at 3 years for GF- and GVHD-II to IV-free survival as event-free survival (EFS) was 56% (46-69) for the entire cohort; 58% (46-72) and 56% (23-88) for LAD-I and LAD-III, respectively. Grade II to IV acute GVHD was a relevant risk factor for death (hazard ratio 3.6; 95% CI 1.4-9.1; P = .006). Patients' age at transplant ≥13 months, transplantation from a nonsibling donor, and any serological cytomegalovirus mismatch in donor-recipient pairs were significantly associated with severe acute GVHD and inferior EFS. The choice of busulfan- or treosulfan-based conditioning, type of GVHD prophylaxis, and serotherapy did not impact overall survival, EFS, or aGVHD. An intrinsic inflammatory component of LAD may contribute to inflammatory complications during allo-HSCT, thus providing the rationale for considering anti-inflammatory therapy pretreatment.
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http://dx.doi.org/10.1182/bloodadvances.2020002185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7805328PMC
January 2021

Arginase 1 IL-10 polymorphonuclear myeloid-derived suppressor cells are elevated in patients with active pemphigus and correlate with an increased Th2/Th1 response.

Exp Dermatol 2021 Jun 23;30(6):782-791. Epub 2021 Feb 23.

Molecular Immunology Charité (MIC), Department of Dermatology, Venereology and Allergology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität of Berlin, and Berlin Institute of Health, Berlin, Germany.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells, which are characterized by their capability to suppress T-cell responses. While MDSCs have been traditionally associated with cancer diseases, their role as regulators of autoimmune diseases is emerging. Pemphigus is a chronic autoimmune blistering skin disease characterized by dysregulated T-cell responses and autoantibody production. The role of MDSCs in pemphigus disease has not been defined yet. The aim of this study was to characterize MDSCs in pemphigus patients and to dissect their relationship with CD4 T-cell subsets and clinical disease assessments. For this purpose, we performed a cross-sectional analysis of 20 patients with pemphigus. Our results indicate that a population of CD66b CD11b polymorphonuclear-like MDSCs (PMN-MDSCs) is expanded in the peripheral blood mononuclear cell fraction of pemphigus patients compared to age-matched healthy donors. These PMN-MDSCs have the capability of suppressing allogeneic T-cell proliferation in vitro and show increased expression of characteristic effector molecules such as arginase I and interleukin-10. We further demonstrate that PMN-MDSCs are especially expanded in patients with active pemphigus, but not in patients in remission. Moreover, MDSC frequencies correlate with an increased Th2/Th1 cell ratio. In conclusion, the identification of a functional PMN-MDSC population suggests a possible role of these cells as regulators of Th cell responses in pemphigus.
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http://dx.doi.org/10.1111/exd.14298DOI Listing
June 2021

A case series of children and young people admitted to a tertiary care hospital in Germany with COVID-19.

BMC Infect Dis 2021 Feb 1;21(1):133. Epub 2021 Feb 1.

Department of Haematology and Oncology, University Children's Hospital Tuebingen, Hoppe-Seyler-Straße 1, 72076, Tuebingen, Germany.

Background: While our knowledge about COVID-19 in adults has rapidly increased, data on the course of disease and outcome in children with different comorbidities is still limited.

Methods: Prospective, observational study at a tertiary care children's hospital in southern Germany. Clinical and virology data from all paediatric patients admitted with SARS-CoV-2 infection at our hospital were prospectively assessed.

Results: Between March and November 2020, 14 patients were admitted with COVID-19. One patient was admitted a second time with COVID-19 6 months after initial disease. Among seven patients with severe underlying comorbidities, three developed multisystem inflammatory syndrome (MIS-C), two were admitted to the paediatric intensive care unit. One patient needed invasive ventilation. Another patient died shortly after discharge of COVID-19-related complications.

Conclusions: While COVID-19 generally causes mild disease in children, severe respiratory illness and MIS-C occur, in some cases with fatal outcome. Children with underlying diseases might be at special risk for severe disease.
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http://dx.doi.org/10.1186/s12879-021-05791-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848863PMC
February 2021

Fulminant mucormycosis during anti-leukemic treatment with blinatumomab in a child: A case report and review of the literature.

Med Mycol Case Rep 2021 Jun 29;32:4-9. Epub 2020 Dec 29.

University Children's Hospital Tuebingen, Department I - General Pediatrics, Hematology/Oncology, Hoppe-Seyler-Str.1, 72076, Tuebingen, Germany.

This is the first published case report of a child with acute lymphatic leukemia developing a fatal mucormycosis during blinatumomab treatment. The patient showed multiple, systemic thromboembolic lesions with ischemia, bleeding and infarction in almost all organs. The child succumbed to increased brain pressure resulting in cerebral herniation. This case particularly illustrates the fulminant progression and huge challenges of diagnosing and treating mucormycosis in children with hemato-oncological diseases during treatment with targeted therapeutic antibodies (blinatumomab).
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http://dx.doi.org/10.1016/j.mmcr.2020.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7806943PMC
June 2021

Prevalence of SARS-CoV-2 Infection in Children and Their Parents in Southwest Germany.

JAMA Pediatr 2021 06;175(6):586-593

Institute of Virology, University Medical Centre and Faculty of Medicine, University of Freiburg, Freiburg im Breisgau, Germany.

Importance: School and daycare closures were enforced as measures to confine the novel coronavirus disease 2019 (COVID-19) pandemic, based on the assumption that young children may play a key role in severe acute respiratory coronavirus 2 (SARS-CoV-2) spread. Given the grave consequences of contact restrictions for children, a better understanding of their contribution to the COVID-19 pandemic is of great importance.

Objective: To describe the rate of SARS-CoV-2 infections and the seroprevalence of SARS-CoV-2 antibodies in children aged 1 to 10 years, compared with a corresponding parent of each child, in a population-based sample.

Design, Setting, And Participants: This large-scale, multicenter, cross-sectional investigation (the COVID-19 BaWü study) enrolled children aged 1 to 10 years and a corresponding parent between April 22 and May 15, 2020, in southwest Germany.

Exposures: Potential exposure to SARS-CoV-2.

Main Outcomes And Measures: The main outcomes were infection and seroprevalence of SARS-CoV-2. Participants were tested for SARS-CoV-2 RNA from nasopharyngeal swabs by reverse transcription-polymerase chain reaction and SARS-CoV-2 specific IgG antibodies in serum by enzyme-linked immunosorbent assays and immunofluorescence tests. Discordant results were clarified by electrochemiluminescence immunoassays, a second enzyme-linked immunosorbent assay, or an in-house Luminex-based assay.

Results: This study included 4964 participants: 2482 children (median age, 6 [range, 1-10] years; 1265 boys [51.0%]) and 2482 parents (median age, 40 [range, 23-66] years; 615 men [24.8%]). Two participants (0.04%) tested positive for SARS-CoV-2 RNA. The estimated SARS-CoV-2 seroprevalence was low in parents (1.8% [95% CI, 1.2-2.4%]) and 3-fold lower in children (0.6% [95% CI, 0.3-1.0%]). Among 56 families with at least 1 child or parent with seropositivity, the combination of a parent with seropositivity and a corresponding child with seronegativity was 4.3 (95% CI, 1.19-15.52) times higher than the combination of a parent who was seronegative and a corresponding child with seropositivity. We observed virus-neutralizing activity for 66 of 70 IgG-positive serum samples (94.3%).

Conclusions And Relevance: In this cross-sectional study, the spread of SARS-CoV-2 infection during a period of lockdown in southwest Germany was particularly low in children aged 1 to 10 years. Accordingly, it is unlikely that children have boosted the pandemic. This SARS-CoV-2 prevalence study, which appears to be the largest focusing on children, is instructive for how ad hoc mass testing provides the basis for rational political decision-making in a pandemic.
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http://dx.doi.org/10.1001/jamapediatrics.2021.0001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823424PMC
June 2021

Adapter chimeric antigen receptor (AdCAR)-engineered NK-92 cells: an off-the-shelf cellular therapeutic for universal tumor targeting.

Oncoimmunology 2020 09 29;9(1):1825177. Epub 2020 Sep 29.

Department of Hematology and Oncology, University Hospital Tuebingen, Children's Hospital, Tuebingen, Germany.

Despite the recent success of CAR T cells targeting CD19 and CD22 in hematological malignancies, the production of CAR T cells still requires an extensive manufacturing process. The well-established NK-92 cell line provides a promising alternative to produce CAR-modified effector cells in a GMP-compliant, cost-effective way. NK-92 can be redirected against a variety of surface antigens by our adapter CAR (AdCAR) system utilizing biotinylated antibodies (bAb) as adapter molecules. Selected bAb were capable of inducing significant AdCAR NK-92-mediated lysis of non-Hodgkin lymphoma (NHL) and mantle-cell lymphoma (MCL) cell lines as well as primary MCL and chronic lymphocytic leukemia (CLL) cells. AdCAR specificity was proven using a JeKo-1 CD19/CD20 knockout antigen-loss model. Moreover, through combinations of bAb, AdCAR NK-92 cells are capable of combatting tumor antigen evasion mechanisms. In conclusion, we successfully generated the AdCAR NK-92 cell line which can be manufactured as an "off-the-shelf, on-demand" product allowing universal and tunable tumor targeting.
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http://dx.doi.org/10.1080/2162402X.2020.1825177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7781805PMC
September 2020

Long-Term Follow-Up After the Application of Mesenchymal Stromal Cells in Children and Adolescents with Steroid-Refractory Graft-Versus-Host Disease.

Stem Cells Dev 2021 03 19;30(5):234-246. Epub 2021 Feb 19.

Division for Pediatric Stem Cell Transplantation and Immunology, Clinic for Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Steroid-refractory graft-versus-host disease (GvHD) is a life-threatening complication after allogeneic hematopoietic stem cell transplantation (alloHSCT). Alternative treatment options are often insufficient. Several studies have proven the efficacy of mesenchymal stromal cells (MSCs) in the treatment of therapy-refractory acute GvHD in adult and pediatric patients. Long-term data in pediatric patients are scarce. In this retrospective analysis, a total of 25 patients with a median age of 10.6 years (range 0.6-22.1 years) who received bone marrow-derived MSCs after alloHSCT for the treatment of steroid-refractory III and IV GvHD were analyzed. The median observation period of the surviving patients was 9.3 years (1.3-12.7 years) after HSCT. Among the 25 patients, 10 (40.0%) died [relapse ( = 3), multiorgan failure ( = 6), cardiorespiratory failure ( = 1)] at median 0.5 years (0.2-2.3 years) after HSCT. Partial response and complete remission (PR, CR) of the GvHD were achieved in 76.0% and 24.0% of the patients, respectively. Transplant-related mortality was 0% in the patients who achieved CR after MSC treatment and 26.3% for those with PR. A median improvement by one intestinal or liver GvHD stage (range 1-4) could be achieved after MSC application. No potentially MSC-related long-term adverse effects, for example, secondary malignancy, were identified. In conclusion, the intravenous application of allogeneic MSCs was safe and proved effective for the treatment of steroid-refractory GvHD. However, larger, prospective, and randomized trials are needed to evaluate these findings.
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http://dx.doi.org/10.1089/scd.2020.0191DOI Listing
March 2021

Establishment and Characterization of a Sclerosing Spindle Cell Rhabdomyosarcoma Cell Line with a Complex Genomic Profile.

Cells 2020 12 11;9(12). Epub 2020 Dec 11.

Department of Orthopaedic Surgery, Eberhard Karls University Tuebingen, 72072 Tuebingen, Germany.

Sclerosing spindle cell rhabdomyosarcoma (SSRMS) is a rare rhabdomyosarcomas (RMS) subtype. Especially cases bearing a myogenic differentiation 1 () mutation are characterized by a high recurrence and metastasis rate, often leading to a fatal outcome. SSRMS cell lines are valuable in vitro models for studying disease mechanisms and for the preclinical evaluation of new therapeutic approaches. In this study, a cell line established from a primary SSRMS tumor of a 24-year-old female after multimodal chemotherapeutic pretreatment has been characterized in detail, including immunohistochemistry, growth characteristics, cytogenetic analysis, mutation analysis, evaluation of stem cell marker expression, differentiation potential, and tumorigenicity in mice. The cell line which was designated SRH exhibited a complex genomic profile, including several translocations and deletions. Array-comparative genomic hybridization (CGH) revealed an overall predominating loss of gene loci. The mesenchymal tumor origin was underlined by the expression of mesenchymal markers and potential to undergo adipogenic and osteogenic differentiation. Despite myogenic marker expression, terminal myogenic differentiation was inhibited, which might be elicited by the hotspot mutation. In vivo tumorigenicity could be confirmed after subcutaneous injection into NOD/SCID/γ mice. Summarized, the SRH cell line is the first adult SSRMS cell line available for preclinical research on this rare RMS subtype.
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http://dx.doi.org/10.3390/cells9122668DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7763666PMC
December 2020

Blinatumomab in pediatric patients with relapsed/refractory B-cell precursor acute lymphoblastic leukemia.

Eur J Haematol 2021 Apr 11;106(4):473-483. Epub 2021 Jan 11.

Department I - General Pediatrics, Hematology/Oncology, Children's Hospital, University Hospital Tübingen, Tübingen, Germany.

Objective: Pediatric patients with relapsed or refractory acute lymphoblastic leukemia have a poor prognosis. We here assess the response rates, adverse events, and long-term follow-up of pediatric patients with relapsed/refractory acute lymphoblastic leukemia receiving blinatumomab.

Methods: Retrospective analysis of a single-center experience with blinatumomab in 38 patients over a period of 10 years.

Results: The median age at onset of therapy was 10 years (1-21 years). Seventy-one percent of patients had undergone at least one hematopoietic stem cell transplantation (HSCT) prior to treatment with blinatumomab. We observed a response to blinatumomab in 13/38 patients (34%). The predominant side effect was febrile reactions, nearly half of the patients developed a cytokine release syndrome. Eight events of neurotoxicity were registered over the 78 cycles (15%). To date, nine patients (24%) are alive and in complete molecular remission. All survivors underwent haploidentical HSCT after treatment with blinatumomab.

Conclusions: Despite heavy pretreatment of most of our patients, severe adverse events were rare and response rates encouraging. Blinatumomab is a valuable bridging salvage therapy for relapsed or refractory patients to a second or even third HSCT.
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http://dx.doi.org/10.1111/ejh.13569DOI Listing
April 2021

CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia.

N Engl J Med 2021 01 5;384(3):252-260. Epub 2020 Dec 5.

From the Sarah Cannon Center for Blood Cancer at the Children's Hospital at TriStar Centennial, Nashville (H.F., J.D.), and St. Jude Children's Research Hospital, Memphis (A.S.) - both in Tennessee; Vertex Pharmaceuticals (D.A., B.K.E., J.L.-H., A.Y.) and Boston University School of Medicine (M.H.S.), Boston, and CRISPR Therapeutics, Cambridge (Y.-S.C., T.W.H., A. Kernytsky, S. Soni) - both in Massachusetts; the University of Milan, Milan (M.D.C.), and Ospedale Pediatrico Bambino Gesù Rome, Sapienza, University of Rome, Rome (F.L.); the University of Regensburg, Regensburg (J. Foell, S.C.), and Children's University Hospital, University of Tübingen, Tübingen (R.H.) - both in Germany; Imperial College Healthcare NHS Trust, St. Mary's Hospital, London (J. de la Fuente); Children's Hospital of Philadelphia, Perelman School of Medicine at the University of Pennsylvania, Philadelphia (S.G.); the University of Athens, Athens (A. Kattamis); BC Children's Hospital, University of British Columbia, Vancouver (A.M.L.), and the Hospital for Sick Children-University of Toronto, Toronto (D.W.) - both in Canada; Columbia University (M.Y.M.) and the Joan and Sanford I. Weill Medical College of Cornell University (S. Sheth), New York; Necker-Enfants Malades Hospital, Assistance Publique-Hôpitaux de Paris, University of Paris, Paris (M.M.); and the University of Illinois at Chicago, Chicago (D.R.).

Transfusion-dependent β-thalassemia (TDT) and sickle cell disease (SCD) are severe monogenic diseases with severe and potentially life-threatening manifestations. BCL11A is a transcription factor that represses γ-globin expression and fetal hemoglobin in erythroid cells. We performed electroporation of CD34+ hematopoietic stem and progenitor cells obtained from healthy donors, with CRISPR-Cas9 targeting the erythroid-specific enhancer. Approximately 80% of the alleles at this locus were modified, with no evidence of off-target editing. After undergoing myeloablation, two patients - one with TDT and the other with SCD - received autologous CD34+ cells edited with CRISPR-Cas9 targeting the same enhancer. More than a year later, both patients had high levels of allelic editing in bone marrow and blood, increases in fetal hemoglobin that were distributed pancellularly, transfusion independence, and (in the patient with SCD) elimination of vaso-occlusive episodes. (Funded by CRISPR Therapeutics and Vertex Pharmaceuticals; ClinicalTrials.gov numbers, NCT03655678 for CLIMB THAL-111 and NCT03745287 for CLIMB SCD-121.).
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http://dx.doi.org/10.1056/NEJMoa2031054DOI Listing
January 2021

Antiemetic Prophylaxis with Fosaprepitant and 5-HT-Receptor Antagonists in Pediatric Patients Undergoing Autologous Hematopoietic Stem Cell Transplantation.

Drug Des Devel Ther 2020 25;14:3915-3927. Epub 2020 Sep 25.

Department of General Pediatrics, Hematology/Oncology, University Children's Hospital Tübingen, Tübingen, Germany.

Background: High-dose myeloablative conditioning prior to autologous hematopoietic stem cell transplantation (autoHSCT) in pediatric patients is usually highly emetogenic. The antiemetic neurokinin-1 receptor antagonist fosaprepitant was safe and effective in children receiving highly emetogenic chemotherapy. Data on fosaprepitant during autoHSCT in children are currently not available.

Methods: A total of 35 consecutive pediatric patients, who received an antiemetic prophylaxis with fosaprepitant (4 mg/kg; single dose, max. 1 x 150 mg/kg BW) and ondansetron (24-hours continuous infusion; 8-32 mg/24h) or granisetron (2 x 40 µg/kg∙d) during highly emetogenic conditioning chemotherapy before autoHSCT were retrospectively analyzed, and their results were compared with a control group comprising 35 consecutive pediatric patients, who received granisetron or ondansetron only. The antiemetic efficacy and the safety of the two prophylaxis regimens were compared with respect to three time periods after the first chemotherapy administration (0-24h, >24-120h, >120-240h).

Results: Clinical adverse events and clinically relevant increases/decreases of laboratory markers were similarly low and did not significantly differ between the two study groups (>0.05). The registered number of vomiting events was significantly higher in the control group in the time periods of 0-24h (64 vs 22 events; <0.01), >24-120h (135 vs 78 events; <0.0001), >120-240h (268 vs 105 events; <0.0001), and the whole observation period 0-240h (467 vs 205 events; <0.0001). The percentage of patients experiencing vomiting was higher in the control group during the time period of >24-120h (100% vs 74.3%) but not the other analyzed time periods (>0.05).

Conclusion: The fosaprepitant-based antiemetic prophylaxis was safe, well tolerated and significantly reduced vomiting in children undergoing highly emetogenic chemotherapy prior to autoHSCT. Prospective randomized trials are necessary to confirm these results.
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http://dx.doi.org/10.2147/DDDT.S260887DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7524181PMC
July 2021

Matched versus Haploidentical Hematopoietic Stem Cell Transplantation as Treatment Options for Primary Immunodeficiencies in Children.

Transplant Cell Ther 2021 01 20;27(1):71.e1-71.e12. Epub 2020 Sep 20.

Department of Pediatrics, Skåne University Hospital, Lund, Sweden.

Primary immunodeficiencies (PIDs) are inherited disorders of the immune system with allogeneic hematopoietic stem cell transplantation (HSCT) as the only curative treatment in some of them. In case an HLA-matched donor is not available, HSCT from a haploidentical family donor may be considered. We compared the outcomes of HSCT from HLA-matched unrelated or related donors (MUDs or MRDs) and mismatched related haploidentical donors (MMRDs) in patients with a variety of PIDs in 2 centers. A total of 44 pediatric patients were evaluated. We reviewed the outcomes of 25 children who underwent transplantation with HLA-matched grafts (MRD, n = 13; MUD, n = 12) and 19 patients receiving haploidentical stem cells. Bone marrow (BM) was transplanted in 85% (MRD) and 75% (MUD) of the matched cohort and peripheral blood stem cells (PBSCs) in 15% (MRD), 25% (MUD), and 100% (MMRD). All but 9 patients (MRD, n = 6; MMRD, n = 3) with severe combined immunodeficiency (SCID) received a chemotherapy-based conditioning regimen. Immune reconstitution of T, B, and natural killer cells was comparable for all groups with an advantage of recipients of MRD grafts in early CD4 reconstitution. However, deaths due to viral infections occurred more often in the haploidentical cohort. The disease-free survival was 91.7% (MRD), 66.7% (MUD), and 62.7% (MMRD), respectively. Grade II to IV acute graft-versus-host disease (GVHD) occurred in 15% (MRD), 8% (MUD), and 21% (MMRD) of the patients. Only 1 patient had severe grade IV GVHD in the MRD group, whereas no grade >II GVHD was observed in the MUD or MMRD cohort. These data indicate that in the absence of a suitable HLA-identical family donor, haploidentical HSCT may be a viable option for patients with life-threatening disease and urgent need of HSCT.
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http://dx.doi.org/10.1016/j.bbmt.2020.09.010DOI Listing
January 2021

Defibrotide for the Treatment of Pediatric Inflammatory Multisystem Syndrome Temporally Associated With Severe Acute Respiratory Syndrome Coronavirus 2 Infection in 2 Pediatric Patients.

J Pediatric Infect Dis Soc 2020 Nov;9(5):622-625

Children's University Hospital, Eberhard Karls University Tuebingen, Tuebingen, Germany.

The pediatric inflammatory multisystem syndrome temporally associated with severe acute respiratory syndrome coronavirus 2 infection is a severe complication of coronavirus disease 2019. Since impaired coagulation and thrombosis/endotheliitis are suspected pathomechanisms, we treated 2 patients with defibrotide, a profibrinolytic, antithrombotic, antiinflammatory oligonucleotide. Symptoms resolved during treatment. Moreover, coagulation parameters indicating hypofibrinolysis and complement activation normalized. The pediatric inflammatory multisystem syndrome temporally associated with severe acute respiratory syndrome coronavirus 2 infection is a severe complication of coronavirus disease 2019. Since impaired coagulation and thrombosis/endotheliitis are suspected pathomechanisms, 2 patients received defibrotide, a profibrinolytic, antithrombotic, antiinflammatory oligonucleotide. Symptoms resolved and hypofibrinolysis/complement activation normalized during treatment.
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http://dx.doi.org/10.1093/jpids/piaa117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7543556PMC
November 2020

Comparative targeting analysis of KLF1, BCL11A, and HBG1/2 in CD34 HSPCs by CRISPR/Cas9 for the induction of fetal hemoglobin.

Sci Rep 2020 06 23;10(1):10133. Epub 2020 Jun 23.

University Children's Hospital. Department of Pediatrics I, Hematology and Oncology, University of Tübingen, Tübingen, Germany.

β-hemoglobinopathies are caused by abnormal or absent production of hemoglobin in the blood due to mutations in the β-globin gene (HBB). Imbalanced expression of adult hemoglobin (HbA) induces strong anemia in patients suffering from the disease. However, individuals with natural-occurring mutations in the HBB cluster or related genes, compensate this disparity through γ-globin expression and subsequent fetal hemoglobin (HbF) production. Several preclinical and clinical studies have been performed in order to induce HbF by knocking-down genes involved in HbF repression (KLF1 and BCL11A) or disrupting the binding sites of several transcription factors in the γ-globin gene (HBG1/2). In this study, we thoroughly compared the different CRISPR/Cas9 gene-disruption strategies by gene editing analysis and assessed their safety profile by RNA-seq and GUIDE-seq. All approaches reached therapeutic levels of HbF after gene editing and showed similar gene expression to the control sample, while no significant off-targets were detected by GUIDE-seq. Likewise, all three gene editing platforms were established in the GMP-grade CliniMACS Prodigy, achieving similar outcome to preclinical devices. Based on this gene editing comparative analysis, we concluded that BCL11A is the most clinically relevant approach while HBG1/2 could represent a promising alternative for the treatment of β-hemoglobinopathies.
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http://dx.doi.org/10.1038/s41598-020-66309-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7311455PMC
June 2020

Comparative analysis of lentiviral gene transfer approaches designed to promote fetal hemoglobin production for the treatment of β-hemoglobinopathies.

Blood Cells Mol Dis 2020 09 29;84:102456. Epub 2020 May 29.

University Children's Clinic Department of Pediatrics I, Hematology and Oncology, University of Tübingen, Germany. Electronic address:

β-Hemoglobinopathies are among the most common single-gene disorders and are caused by different mutations in the β-globin gene. Recent curative therapeutic approaches for these disorders utilize lentiviral vectors (LVs) to introduce a functional copy of the β-globin gene into the patient's hematopoietic stem cells. Alternatively, fetal hemoglobin (HbF) can reduce or even prevent the symptoms of disease when expressed in adults. Thus, induction of HbF by means of LVs and other molecular approaches has become an alternative treatment of β-hemoglobinopathies. Here, we performed a head-to-head comparative analysis of HbF-inducing LVs encoding for: 1) IGF2BP1, 2) miRNA-embedded shRNA (shmiR) sequences specific for the γ-globin repressor protein BCL11A, and 3) γ-globin gene. Furthermore, two novel baboon envelope proteins (BaEV)-LVs were compared to the commonly used vesicular-stomatitis-virus glycoprotein (VSV-G)-LVs. Therapeutic levels of HbF were achieved for all VSV-G-LV approaches, from a therapeutic level of 20% using γ-globin LVs to 50% for both IGF2BP1 and BCL11A-shmiR LVs. Contrarily, BaEV-LVs conferred lower HbF expression with a peak level of 13%, however, this could still ameliorate symptoms of disease. From this thorough comparative analysis of independent HbF-inducing LV strategies, we conclude that HbF-inducing VSV-G-LVs represent a promising alternative to β-globin gene addition for patients with β-hemoglobinopathies.
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http://dx.doi.org/10.1016/j.bcmd.2020.102456DOI Listing
September 2020
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