Karl Welte

Granulocyte and granulocyte-macrophage colony-stimulating factors for treatment of neutropenia in glycogen storage disease type ib

Two children with glycogen storage disease type Ib associated with numerous recurrent bacterial infections as a result of neutropenia and neutrophil dysfunction were treated with recombinant human granulocyte colony-stimulating factor (G-CSF). One of the two patients was previously treated with recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF); therapy had to be discontinued because of severe local side effects. Both colony-stimulating factors at dosages of 3 and 8 micrograms/kg/per day, respectively, increased the average neutrophil counts from less than 300 cells/microliters to more than 1200 cells/microliters. Two subpopulations of neutrophils could be identified by their capacity to produce H2O2: one subpopulation generated H2O2 normally and a second was defective in H2O2 production. The doses of G-CSF effectively enhanced and maintained that subpopulation of neutrophils which produced normal amounts of H2O2. Moreover, these colony-stimulating factor-induced neutrophils demonstrated effective phagocytosis of zymosan particles and killing of staphylococci. Chemotaxis was decreased and could not be normalized by treatment with G-CSF. We conclude that maintenance treatment with G-CSF improved the quality of life in both patients: The number and severity of bacterial infections decreased markedly during treatment. Long-term treatment with G-CSF (12 and 10 months, respectively) was well tolerated, and no adverse clinical events were observed.

Gene correction of HAX1 reversed Kostmann disease phenotype in patient-specific induced pluripotent stem cells

Severe congenital neutropenia (SCN, Kostmann disease) is a heritable disorder characterized by a granulocytic maturation arrest. Biallelic mutations in HCLS1 associated protein X-1 (HAX1) are frequently detected in affected individuals, including those of the original pedigree described by Kostmann in 1956. To date, no faithful animal model has been established to study SCN mediated by HAX1 deficiency. Here we demonstrate defective neutrophilic differentiation and compensatory monocyte overproduction from patient-derived induced pluripotent stem cells (iPSCs) carrying the homozygous HAX1W44X nonsense mutation. Targeted correction of the HAX1 mutation using the CRISPR-Cas9 system and homologous recombination rescued neutrophil differentiation and reestablished an HAX1 and HCLS1-centered transcription network in immature myeloid progenitors, which is involved in the regulation of apoptosis, apoptotic mitochondrial changes, and myeloid differentiation. These findings made in isogenic iPSC-derived myeloid cells highlight the complex transcriptional changes underlying Kostmann disease. Thus, we show that patient-derived HAX1W44X -iPSCs recapitulate the Kostmann disease phenotype in vitro and confirm HAX1 mutations as the disease-causing monogenic lesion. Finally, our study paves the way for nonvirus-based gene therapy approaches in SCN.

GM-CSF treatment is not effective in congenital neutropenia patients due to its inability to activate NAMPT signaling.

Severe congenital neutropenia (CN) is a bone marrow failure syndrome characterized by an absolute neutrophil count (ANC) below 500xa0cells/μL and recurrent, life-threatening bacterial infections. Treatment with granulocyte colony-stimulating factor (G-CSF) increases the ANC in the majority of CN patients. In contrary, granulocyte-monocyte colony-stimulating factor (GM-CSF) fails to increase neutrophil numbers in CN patients in vitro and in vivo, suggesting specific defects in signaling pathways downstream of GM-CSF receptor. Recently, we detected that G-CSF induces granulopoiesis in CN patients by hyperactivation of nicotinamide phosphoribosyl transferase (NAMPT)/Sirtuin 1 signaling in myeloid cells. Here, we demonstrated that, in contrast to G-CSF, GM-CSF failed to induce NAMPT-dependent granulopoiesis in CN patients. We further identified NAMPT signaling as an essential downstream effector of the GM-CSF pathway in myelopoiesis.

Thrombopoietin induces hematopoiesis from mouse ES cells via HIF‐1α–dependent activation of a BMP4 autoregulatory loop

Understanding the molecular mechanisms underlying hematopoietic differentiation of embryonic stem (ES) cells may help to ascertain the conditions for the in vitro generation of hematopoietic cells. Previously, we found that patients with congenital amegakaryocytic thrombocytopenia (CAMT), who develop pancytopenia early after birth, harbor mutations within the thrombopoietin (TPO) receptor, c‐MPL. This knowledge, together with observations in vitro and in vivo, suggests that TPO/c‐MPL signaling promotes early hematopoiesis. However, the mechanisms underlying TPO signaling are not fully elucidated. Here, we describe a direct connection between TPO and bone morphogenetic protein 4 (BMP4) signaling pathways in determining the hematopoietic fate of ES cells. Morphogen BMP4 is known to induce early hematopoietic differentiation of ES cells. Treatment of ES cells with TPO induced the autocrine production of BMP4 with concomitant upregulation of the BMP receptor BMPR1A, phosphorylation of SMAD1, 5, 8, and activation of specific BMP4 target genes; this was mediated by TPO‐dependent binding of transcription factor HIF‐1α to the BMP4 gene promoter. Treatment of ES cells with the BMP antagonist noggin substantially reduced TPO‐dependent hematopoietic differentiation of ES cells. Thus, our findings contribute to the establishment of techniques for generating hematopoietic cells from ES cells.

Angeborene Störungen der Blutbildung

ZusammenfassungAngeborene Störungen der Blutbildung umfassen eine Gruppe seltener Erkrankungen, die durch eine Verminderung reifer Blutzellen (Erythrozyten, Granulozyten, Thrombozyten) gekennzeichnet sind. Zu diesen Erkrankungen gehören die angeborene aplastische Anämie (Fanconi-Anämie), kongenitale hypoplastische Anämie (Diamond-Blackfan-Anämie), kongenitale Neutropenien (Kostmann-Syndrom, zyklische Neutropenie, Shwachman-Diamond-Syndrom und viele andere) sowie kongenitale Thrombozytopenien (TAR-Syndrom, amegakaryozytäre Thrombozytopenie). Insgesamt ist in Deutschland von einer Prävalenz dieser Erkrankungen von 10/1.000.000 Kindern und Jugendlichen auszugehen. Trotz ihrer Seltenheit ist ihr Verständnis für die Erforschung der normalen und pathologischen Hämatopoese von immenser Bedeutung. Zu diesem Zweck sind die Dokumentation dieser seltenen Erkrankungen in Patientenregistern und der Zusammenschluss von Behandlungszentren in Netzwerken wichtig. Im Folgenden soll dies am Beispiel der angeborenen Neutropenien beschrieben werden: Bis in die 1980er-Jahre war eine Klassifikation der angeborenen Neutropenien ausschließlich klinisch möglich, es existierten nur wenige Fallbeschreibungen. Daher wurden die unterschiedlichen Formen unter dem Sammelbegriff schwere kongenitale Neutropenie zusammengefasst. Erst durch den Aufbau eines internationalen Expertennetzwerkes und die Langzeitdokumentation von Krankheitsverläufen in einer gemeinsamen Datenbank konnten statistisch tragfähige Daten zu Therapieansprechen, Begleiterkrankungen und Langzeitprognose gesammelt werden. Die enge Kooperation mit Wissenschaftlern führte letztlich zur Charakterisierung genetisch unterschiedlicher Erkrankungen mit gemeinsamen Pathomechanismen.AbstractCongenital bone marrow failure syndromes are rare diseases characterised by a reduction of mature blood cells (erythrocytes, platelets, neutrophils). Examples of such disorders include congenital aplastic anemia (Fanconi anemia), congenital hypoplastic anemia (Diamond-Blackfan anemia), congenital neutropenias (Kostmann syndrome, cyclic neutropenia, Shwachman-Diamond syndrome and others), and congenital thrombocytopenias (TAR syndrome, amegacaryocytic thrombocytopenia). In Germany the prevalence of congenital bone marrow failure syndromes can be estimated to be 10/1,000,000 children and adolescents. Although rare, these diseases contributed significantly to the current knowledge on normal haematopoiesis. The documentation of rare diseases by patient registries and the cooperation of clinical centres within networks are most important for the resolution of such disorders. In the following, congenital neutropenia will be presented as an example: Until the 1980s congenital neutropenia could only be classified clinically. Few cases had been reported in the literature. All subtypes were therefore collected under the general term congenital neutropenia. The establishment of an international network of experts and the long-term documentation of the courses of disease in a common database allowed for statistically workable data in response to therapy, secondary diagnoses and the long-term prognosis. A close cooperation with scientists finally led to the characterisation of genetically different disorders with common pathomechanisms.

Angeborene Störungen der Blutbildung@@@Congenital bone marrow failure syndromes. The last 20 years by the example of congenital neutropenia: Die Entwicklung der letzten 20 Jahre am Beispiel der kongenitalen Neutropenie

ZusammenfassungAngeborene Störungen der Blutbildung umfassen eine Gruppe seltener Erkrankungen, die durch eine Verminderung reifer Blutzellen (Erythrozyten, Granulozyten, Thrombozyten) gekennzeichnet sind. Zu diesen Erkrankungen gehören die angeborene aplastische Anämie (Fanconi-Anämie), kongenitale hypoplastische Anämie (Diamond-Blackfan-Anämie), kongenitale Neutropenien (Kostmann-Syndrom, zyklische Neutropenie, Shwachman-Diamond-Syndrom und viele andere) sowie kongenitale Thrombozytopenien (TAR-Syndrom, amegakaryozytäre Thrombozytopenie). Insgesamt ist in Deutschland von einer Prävalenz dieser Erkrankungen von 10/1.000.000 Kindern und Jugendlichen auszugehen. Trotz ihrer Seltenheit ist ihr Verständnis für die Erforschung der normalen und pathologischen Hämatopoese von immenser Bedeutung. Zu diesem Zweck sind die Dokumentation dieser seltenen Erkrankungen in Patientenregistern und der Zusammenschluss von Behandlungszentren in Netzwerken wichtig. Im Folgenden soll dies am Beispiel der angeborenen Neutropenien beschrieben werden: Bis in die 1980er-Jahre war eine Klassifikation der angeborenen Neutropenien ausschließlich klinisch möglich, es existierten nur wenige Fallbeschreibungen. Daher wurden die unterschiedlichen Formen unter dem Sammelbegriff schwere kongenitale Neutropenie zusammengefasst. Erst durch den Aufbau eines internationalen Expertennetzwerkes und die Langzeitdokumentation von Krankheitsverläufen in einer gemeinsamen Datenbank konnten statistisch tragfähige Daten zu Therapieansprechen, Begleiterkrankungen und Langzeitprognose gesammelt werden. Die enge Kooperation mit Wissenschaftlern führte letztlich zur Charakterisierung genetisch unterschiedlicher Erkrankungen mit gemeinsamen Pathomechanismen.AbstractCongenital bone marrow failure syndromes are rare diseases characterised by a reduction of mature blood cells (erythrocytes, platelets, neutrophils). Examples of such disorders include congenital aplastic anemia (Fanconi anemia), congenital hypoplastic anemia (Diamond-Blackfan anemia), congenital neutropenias (Kostmann syndrome, cyclic neutropenia, Shwachman-Diamond syndrome and others), and congenital thrombocytopenias (TAR syndrome, amegacaryocytic thrombocytopenia). In Germany the prevalence of congenital bone marrow failure syndromes can be estimated to be 10/1,000,000 children and adolescents. Although rare, these diseases contributed significantly to the current knowledge on normal haematopoiesis. The documentation of rare diseases by patient registries and the cooperation of clinical centres within networks are most important for the resolution of such disorders. In the following, congenital neutropenia will be presented as an example: Until the 1980s congenital neutropenia could only be classified clinically. Few cases had been reported in the literature. All subtypes were therefore collected under the general term congenital neutropenia. The establishment of an international network of experts and the long-term documentation of the courses of disease in a common database allowed for statistically workable data in response to therapy, secondary diagnoses and the long-term prognosis. A close cooperation with scientists finally led to the characterisation of genetically different disorders with common pathomechanisms.