RESUMO
Aberrantly expressed cytokines in the bone marrow (BM) niche are increasingly recognized as critical mediators of survival and expansion of leukemic stem cells. To identify regulators of primitive chronic myeloid leukemia (CML) cells, we performed a high-content cytokine screen using primary CD34+ CD38low chronic phase CML cells. Out of the 313 unique human cytokines evaluated, 11 were found to expand cell numbers ≥2-fold in a 7-day culture. Focusing on novel positive regulators of primitive CML cells, the myostatin antagonist myostatin propeptide gave the largest increase in cell expansion and was chosen for further studies. Herein, we demonstrate that myostatin propeptide expands primitive CML and normal BM cells, as shown by increased colony-forming capacity. For primary CML samples, retention of CD34-expression was also seen after culture. Furthermore, we show expression of MSTN by CML mesenchymal stromal cells, and that myostatin propeptide has a direct and instant effect on CML cells, independent of myostatin, by demonstrating binding of myostatin propeptide to the cell surface and increased phosphorylation of STAT5 and SMAD2/3. In summary, we identify myostatin propeptide as a novel positive regulator of primitive CML cells and corresponding normal hematopoietic cells.
Assuntos
Células-Tronco Hematopoéticas , Leucemia Mielogênica Crônica BCR-ABL Positiva , Antígenos CD34 , Medula Óssea , Células Cultivadas , Citocinas , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Miostatina/genéticaRESUMO
Chronic myeloid leukemia (CML) is currently treated with tyrosine kinase inhibitors, but these do not effectively eliminate the CML stem cells. As a consequence, CML stem cells persist and cause relapse in most patients upon drug discontinuation. Furthermore, no effective therapy exists for the advanced stages of the disease. Interleukin-1 receptor accessory protein (IL1RAP; IL1R3) is a coreceptor of interleukin-1 receptor type 1 and has been found upregulated on CML stem cells. Here, we show that primitive (CD34+CD38-) CML cells, in contrast to corresponding normal cells, express a functional interleukin-1 (IL-1) receptor complex and respond with NF-κB activation and marked proliferation in response to IL-1. IL1RAP antibodies that inhibit IL-1 signaling could block these effects. In vivo administration of IL1RAP antibodies in mice transplanted with chronic and blast phase CML cells resulted in therapeutic effects mediated by murine effector cells. These results provide novel insights into the role of IL1RAP in CML and a strong rationale for the development of an IL1RAP antibody therapy to target residual CML stem cells.
Assuntos
Anticorpos Antineoplásicos/farmacologia , Proteína Acessória do Receptor de Interleucina-1/antagonistas & inibidores , Interleucina-1/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Proteínas de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Animais , Feminino , Humanos , Proteína Acessória do Receptor de Interleucina-1/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Masculino , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Tyrosine kinase inhibitors (TKIs) are highly effective for the treatment of chronic myeloid leukemia (CML), but very few patients are cured. The major drawbacks regarding TKIs are their low efficacy in eradicating the leukemic stem cells responsible for disease maintenance and relapse upon drug cessation. Herein, we performed ribonucleic acid sequencing of flow-sorted primitive (CD34+CD38low) and progenitor (CD34+ CD38+) chronic phase CML cells, and identified transcriptional upregulation of 32 cell surface molecules relative to corresponding normal bone marrow cells. Focusing on novel markers with increased expression on primitive CML cells, we confirmed upregulation of the scavenger receptor CD36 and the leptin receptor by flow cytometry. We also delineate a subpopulation of primitive CML cells expressing CD36 that is less sensitive to imatinib treatment. Using CD36 targeting antibodies, we show that the CD36 positive cells can be targeted and killed by antibody-dependent cellular cytotoxicity. In summary, CD36 defines a subpopulation of primitive CML cells with decreased imatinib sensitivity that can be effectively targeted and killed using an anti-CD36 antibody.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Antígenos CD36/genética , Mesilato de Imatinib/farmacologia , Leucemia Mieloide de Fase Crônica/imunologia , Anticorpos Antineoplásicos/uso terapêutico , Antígenos de Neoplasias/imunologia , Antígenos CD36/imunologia , Humanos , Mesilato de Imatinib/uso terapêutico , Leucemia Mieloide de Fase Crônica/tratamento farmacológico , Leucemia Mieloide de Fase Crônica/patologia , Análise de Sequência de RNA , Células Tumorais Cultivadas , Regulação para CimaRESUMO
Acute myeloid leukemia (AML) is associated with a poor survival rate, and there is an urgent need for novel and more efficient therapies, ideally targeting AML stem cells that are essential for maintaining the disease. The interleukin 1 receptor accessory protein (IL1RAP; IL1R3) is expressed on candidate leukemic stem cells in the majority of AML patients, but not on normal hematopoietic stem cells. We show here that monoclonal antibodies targeting IL1RAP have strong antileukemic effects in xenograft models of human AML. We demonstrate that effector-cell-mediated killing is essential for the observed therapeutic effects and that natural killer cells constitute a critical human effector cell type. Because IL-1 signaling is important for the growth of AML cells, we generated an IL1RAP-targeting antibody capable of blocking IL-1 signaling and show that this antibody suppresses the proliferation of primary human AML cells. Hence, IL1RAP can be efficiently targeted with an anti-IL1RAP antibody capable of both achieving antibody-dependent cellular cytotoxicity and blocking of IL-1 signaling as modes of action. Collectively, these results provide important evidence in support of IL1RAP as a target for antibody-based treatment of AML.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Proteína Acessória do Receptor de Interleucina-1/antagonistas & inibidores , Leucemia Mieloide Aguda/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Animais , Citotoxicidade Celular Dependente de Anticorpos , Divisão Celular , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Humanos , Interleucina-1/antagonistas & inibidores , Proteína Acessória do Receptor de Interleucina-1/imunologia , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas de Neoplasias/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
IL1RAP, a co-receptor for interleukin (IL)-1 and IL-33 receptors, was previously found to be highly upregulated on candidate chronic myeloid leukemia stem cells, allowing for leukemia-selective killing using IL1RAP-targeting antibodies. We analyzed IL1RAP expression in a consecutive series of 29 patients with acute myeloid leukemia (AML) and, based on the level of expression in mononuclear cells (MNCs), we divided the samples into 3 groups: IL1RAP low (n = 6), IL1RAP intermediate (n = 11), and IL1RAP high (n = 12). Within the CD34+CD38- population, the intermediate and high groups expressed higher levels of IL1RAP than did corresponding normal cells. With the aim to target AML stem cells, an anti-IL1RAP monoclonal antibody was generated followed by isotype switching for improved antibody-dependent, cell-mediated cytotoxicity activity. Using this antibody, we achieved selective killing of AML MNC, CD34+CD38+, and CD34+CD38- cells. Our findings demonstrate that IL1RAP is a promising new therapeutic target in AML.
Assuntos
Anticorpos/farmacologia , Citotoxicidade Imunológica/efeitos dos fármacos , Proteína Acessória do Receptor de Interleucina-1/imunologia , Leucemia Mieloide Aguda/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Especificidade de Anticorpos , Células Cultivadas , Feminino , Humanos , Imunoterapia/métodos , Proteína Acessória do Receptor de Interleucina-1/antagonistas & inibidores , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Células-Tronco Neoplásicas/patologiaRESUMO
4-1BB (CD137) is an activation-induced costimulatory receptor that regulates immune responses of activated CD8 T and natural killer cells, by enhancing proliferation, survival, cytolytic activity, and IFNγ production. The ability to induce potent antitumor activity by stimulating 4-1BB on tumor-specific cytotoxic T cells makes 4-1BB an attractive target for designing novel immuno-oncology therapeutics. To minimize systemic immune toxicities and enhance activity at the tumor site, we have developed a novel bispecific antibody that stimulates 4-1BB function when co-engaged with the tumor-associated antigen 5T4. ALG.APV-527 was built on the basis of the ADAPTIR bispecific platform with optimized binding domains to 4-1BB and 5T4 originating from the ALLIGATOR-GOLD human single-chain variable fragment library. The epitope of ALG.APV-527 was determined to be located at domain 1 and 2 on 4-1BB using X-ray crystallography. As shown in reporter and primary cell assays in vitro, ALG.APV-527 triggers dose-dependent 4-1BB activity mediated only by 5T4 crosslinking. In vivo, ALG.APV-527 demonstrates robust antitumor responses, by inhibiting growth of established tumors expressing human 5T4 followed by a long-lasting memory immune response. ALG.APV-527 has an antibody-like half-life in cynomolgus macaques and was well tolerated at 50.5 mg/kg. ALG.APV-527 is uniquely designed for 5T4-conditional 4-1BB-mediated antitumor activity with potential to minimize systemic immune activation and hepatotoxicity while providing efficacious tumor-specific responses in a range of 5T4-expressing tumor indications as shown by robust activity in preclinical in vitro and in vivo models. On the basis of the combined preclinical dataset, ALG.APV-527 has potential as a promising anticancer therapeutic for the treatment of 5T4-expressing tumors.
Assuntos
Anticorpos Biespecíficos , Neoplasias , Anticorpos de Cadeia Única , Humanos , Anticorpos Biespecíficos/farmacologia , Anticorpos Biespecíficos/uso terapêutico , Antígenos de Neoplasias , Linfócitos T , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral , Ligante 4-1BB/metabolismoRESUMO
Understanding the in vivo regulation of hematopoietic stem cells (HSCs) will be critical to identifying key factors involved in the regulation of HSC self-renewal and differentiation. The niche (microenvironment) in which HSCs reside has recently regained attention accompanied by a dramatic increase in the understanding of the cellular constituents of the bone marrow HSC niche. The use of sophisticated genetic models allowing modulation of specific lineages has demonstrated roles for mesenchymal-derived elements such as osteoblasts and adipocytes, vasculature, nerves, and a range of hematopoietic progeny of the HSC as being participants in the regulation of the bone marrow microenvironment. Whilst providing significant insight into the cellular composition of the niche, is it possible to manipulate any given cell lineage in vivo without impacting, knowingly or unknowingly, on those that remain?
Assuntos
Células-Tronco Hematopoéticas/citologia , Nicho de Células-Tronco/citologia , Animais , Células da Medula Óssea/citologia , Humanos , CamundongosRESUMO
Infantile malignant osteopetrosis is a devastating disorder of early childhood that is frequently fatal and for which there are only limited therapeutic options. Gene therapy utilizing autologous hematopoietic stem and progenitor cells represents a potentially advantageous therapeutic alternative for this multisystemic disease. Gene therapy can be performed relatively rapidly following diagnosis, will not result in graft versus host disease, and may also have potential for reduced incidences of other transplant-related complications. In this review, we have summarized the past sixteen years of research aimed at developing a gene therapy for infantile malignant osteopetrosis; these efforts have culminated in the first clinical trial employing lentiviral-mediated delivery of TCIRG1 in autologous hematopoietic stem and progenitor cells.
RESUMO
Dysfunction in or lack of osteoclasts result in osteopetrosis, a group of rare but often severe, genetic disorders characterized by an increase in bone mass, skeletal malformations and bone marrow failure that may be fatal. Several of the underlying defects have lately been characterized in humans and in animal disease models. In humans, these defects often involve mutations in genes expressing proteins involved in the acidification of the osteoclast sub-cellular compartment, a process necessary for proper bone resorption. So far, the only cure for children with severe osteopetrosis is allogeneic hematopoietic stem cell transplantation (SCT). However, the characterization of the genetic defects opens up the possibility for gene replacement therapy as an alternative to SCT. Recently, gene therapy targeting hematopoietic stem cells (HSC) in a mouse model of infantile malignant osteopetrosis was shown to correct many aspects of the disease. Here we review important aspects of this group of diseases and discuss the prospects for development of gene therapy of osteopetrosis.
Assuntos
Terapia Genética/tendências , Osteopetrose/genética , Osteopetrose/terapia , Animais , Remodelação Óssea/efeitos dos fármacos , Remodelação Óssea/genética , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Camundongos , Osteoclastos/efeitos dos fármacos , Osteoclastos/patologia , Osteopetrose/patologiaRESUMO
Lack of or dysfunction in osteoclasts result in osteopetrosis, a group of rare but often severe, genetic disorders affecting skeletal tissue. Increase in bone mass results in skeletal malformation and bone marrow failure that may be fatal. Many of the underlying defects have lately been characterized in humans and in animal models of the disease. In humans, these defects often involve mutations in genes expressing proteins involved in the acidification of the osteoclast resorption compartment, a process necessary for proper bone degradation. So far, the only cure for children with severe osteopetrosis is allogeneic hematopoietic stem cell (HSC) transplantation but without a matching donor this form of therapy is far from optimal. The characterization of the genetic defects opens up the possibility for gene replacement therapy as an alternative. Accordingly, HSC-targeted gene therapy in a mouse model of infantile malignant osteopetrosis was recently shown to correct many aspects of the disease.
Assuntos
Osteopetrose/etiologia , Osteopetrose/terapia , Animais , Anidrase Carbônica II/deficiência , Canais de Cloreto/deficiência , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas , Humanos , Lactente , Recém-Nascido , Camundongos , Mutação , Osteoclastos/fisiologia , Osteopetrose/fisiopatologia , Bombas de Próton/deficiênciaRESUMO
Several attempts have been made to model chronic myeloid leukemia (CML) in a xenograft setting but expansion of human myeloid cells in immunodeficient mice has proven difficult to achieve. Lack of cross-reacting cytokines in the microenvironment of the mice has been proposed as a potential reason. In this study we have used NOD/SCID IL2-receptor gamma deficient mice expressing human SCF, IL-3 and GM-CSF (NSGS mice), that should be superior in supporting human, and particularly, myeloid cell engraftment, to expand BCR-ABL1 expressing human cells in order to model CML. NSGS mice transplanted with BCR-ABL1 expressing cells became anemic and had to be sacrificed due to illness, however, this was not accompanied by an expansion of human myeloid cells but rather we observed a massive expansion of human T-cells and macrophages/histiocytes. Importantly, control human cells without BCR-ABL1 expression elicited a similar reaction, although with a slight delay of disease induction, suggesting that while BCR-ABL1 contributes to the inflammatory reaction, the presence of normal human hematopoietic cells is detrimental for NSGS mice.
Assuntos
Citocinas/genética , Sangue Fetal/citologia , Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Animais , Modelos Animais de Doenças , Feminino , Sangue Fetal/transplante , Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Humanos , Interleucina-3/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Transdução Genética , Transplante HeterólogoRESUMO
Fusion genes are potent driver mutations in cancer. In this study, we delineate the fusion gene landscape in a consecutive series of 195 paediatric B-cell precursor acute lymphoblastic leukaemia (BCP ALL). Using RNA sequencing, we find in-frame fusion genes in 127 (65%) cases, including 27 novel fusions. We describe a subtype characterized by recurrent IGH-DUX4 or ERG-DUX4 fusions, representing 4% of cases, leading to overexpression of DUX4 and frequently co-occurring with intragenic ERG deletions. Furthermore, we identify a subtype characterized by an ETV6-RUNX1-like gene-expression profile and coexisting ETV6 and IKZF1 alterations. Thus, this study provides a detailed overview of fusion genes in paediatric BCP ALL and adds new pathogenetic insights, which may improve risk stratification and provide therapeutic options for this disease.
Assuntos
Rearranjo Gênico/genética , Proteínas de Homeodomínio/genética , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Processamento Alternativo/genética , Criança , Quebra Cromossômica , Análise por Conglomerados , Estudos de Coortes , Análise Mutacional de DNA , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Análise de Componente PrincipalRESUMO
Skeletal-related events resulting from accelerated bone loss are common complications in patients treated for a range of cancers. However, the mechanisms and rate of bone loss after myelosuppression are unclear. We, therefore, investigated this in mice and humans. We treated mice with different myelosuppressive therapies (chemotherapy or irradiation with or without transplantation) and studied their effects on bone structure. Myelosuppression of mice rapidly caused an increase in bone resorption that was not matched by bone formation. The resultant significant and persistent bone loss early after therapy was associated with increased inflammatory cytokines, in particular, monocyte chemoattractant protein 1 (MCP1). Therapy-induced bone loss was prevented with a single dose of the bisphosphonate zoledronic acid (ZA), administered before myelosuppression. Importantly, ZA treatment of mice did not impair hematopoiesis, including hematopoietic stem cell function. Furthermore, examination of serum from patients before and after autologous or allogeneic stem cell transplantion (SCT) revealed altered levels of bone turnover markers and elevated inflammatory cytokines. MCP1 levels in serum obtained between days 7 and 14 post-SCT positively correlated with bone loss observed at 100 days after allogeneic SCT. Similar to that observed in our studies in mice, the bone loss was long term, persisting at 12 months post-SCT. Furthermore, patients who received chemotherapy less than 100 days before SCT had significantly more bone loss at the hip. In these patients, serum levels of MCP1, but not routine biomarkers of bone turnover, including C-terminal cross-linking telopeptide of type-1 collagen (ß-CTx), positively correlated with their bone loss. Hence, myelosuppressive therapies increase inflammation and directly contribute to bone loss. Administration of an osteoclast inhibitor before the initiation of cancer therapy is likely to have the best outcome in preventing bone loss in patients with cancer.
Assuntos
Antineoplásicos/efeitos adversos , Reabsorção Óssea/induzido quimicamente , Reabsorção Óssea/metabolismo , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Animais , Densidade Óssea/efeitos dos fármacos , Remodelação Óssea/efeitos dos fármacos , Reabsorção Óssea/diagnóstico por imagem , Reabsorção Óssea/patologia , Quimiocina CCL2/sangue , Difosfonatos/farmacologia , Difosfonatos/uso terapêutico , Hematopoese/efeitos dos fármacos , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Inflamação/patologia , Camundongos Endogâmicos C57BL , Transplante de Células-Tronco , Transplante Homólogo , Microtomografia por Raio-X , Ácido ZoledrônicoRESUMO
The gp130 receptor and its binding partners play a central role in cytokine signalling. Ciliary neurotrophic factor (CNTF) is one of the cytokines that signals through the gp130 receptor complex. CNTF has previously been shown to be a negative regulator of trabecular bone remodelling and important for motor neuron development. Since haematopoietic cell maintenance and differentiation is dependent on the bone marrow (BM) microenvironment, where cells of the osteoblastic lineage are important regulators, we hypothesised that CNTF may also have important roles in regulating haematopoiesis. Analysis of haematopoietic parameters in male and female Cntf(-/-) mice at 12 and 24 weeks of age revealed altered B lymphopoiesis. Strikingly, the B lymphocyte phenotype differed based on sex, age and also the BM microenvironment in which the B cells develop. When BM cells from wildtype mice were transplanted into Cntf(-/-) mice, there were minimal effects on B lymphopoiesis or bone parameters. However, when Cntf(-/-) BM cells were transplanted into a wildtype BM microenvironment, there were changes in both haematopoiesis and bone parameters. Our data reveal that haematopoietic cell-derived CNTF has roles in regulating BM B cell lymphopoiesis and both trabecular and cortical bone, the latter in a sex-dependent manner.
Assuntos
Linfócitos B/metabolismo , Diferenciação Celular/genética , Fator Neurotrófico Ciliar/genética , Hematopoese/genética , Animais , Linfócitos B/citologia , Células da Medula Óssea/metabolismo , Remodelação Óssea/genética , Microambiente Celular/genética , Fator Neurotrófico Ciliar/metabolismo , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/metabolismo , Feminino , Ativação Linfocitária/genética , Masculino , Camundongos , Camundongos Transgênicos , Transdução de Sinais/genéticaRESUMO
Infantile malignant osteopetrosis (IMO) is a rare, lethal, autosomal recessive disorder characterized by non-functional osteoclasts. More than 50% of the patients have mutations in the TCIRG1 gene, encoding for a subunit of the osteoclast proton pump. The aim of this study was to restore the resorptive function of IMO osteoclasts by lentiviral mediated gene transfer of the TCIRG1 cDNA. CD34(+) cells from peripheral blood of five IMO patients and from normal cord blood were transduced with lentiviral vectors expressing TCIRG1 and GFP under a SFFV promoter, expanded in culture and differentiated on bone slices to mature osteoclasts. qPCR analysis and western blot revealed increased mRNA and protein levels of TCIRG1, comparable to controls. Vector corrected IMO osteoclasts generated increased release of Ca(2+) and bone degradation product CTX-I into the media as well as increased formation of resorption pits in the bone slices, while non-corrected IMO osteoclasts failed to resorb bone. Resorption was approximately 70-80% of that of osteoclasts generated from cord blood. Furthermore, transduced CD34(+) cells successfully engrafted in NSG-mice. In conclusion we provide the first evidence of lentiviral-mediated correction of a human genetic disease affecting the osteoclastic lineage.
Assuntos
Antígenos CD34/metabolismo , Lentivirus/genética , Osteopetrose/genética , Osteopetrose/terapia , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Células Cultivadas , Criança , Pré-Escolar , Feminino , Citometria de Fluxo , Humanos , Immunoblotting , Lactente , Recém-Nascido , Masculino , Camundongos , Camundongos SCID , Osteoclastos/citologia , Osteoclastos/metabolismo , Reação em Cadeia da PolimeraseRESUMO
The bone marrow (BM) is contained within the bone cavity and is the main site of hematopoiesis, the continuous development of blood cells from immature hematopoietic stem and progenitor cells. The bone marrow consists of developing hematopoietic cells and non-hematopoietic cells, the latter collectively termed the bone marrow microenvironment. These non-hematopoietic cells include cells of the osteoblast lineage, adipocytes and endothelial cells. For many years these bone marrow microenvironment cells were predicted to play active roles in regulating hematopoiesis, and recent studies have confirmed such roles. Importantly, more recent data has indicated that cells of the BM microenvironment may also contribute to hematopoietic diseases. In this review we provide an overview of the roles of the data suggesting that the cells of the bone marrow microenvironment may play an active role in the initiation and progression of hematopoietic malignancy.
Assuntos
Células da Medula Óssea/fisiologia , Medula Óssea/fisiologia , Neoplasias Hematológicas/patologia , Leucemia/patologia , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Células da Medula Óssea/citologia , Osso e Ossos/fisiologia , Humanos , Modelos Animais , Células-TroncoRESUMO
Osteopetrosis caused by defective acid secretion by the osteoclast, is characterized by defective bone resorption, increased osteoclast numbers, while bone formation is normal or increased. In contrast the bones are of poor quality, despite this uncoupling of formation from resorption.To shed light on the effect of uncoupling in adult mice with respect to bone strength, we transplanted irradiated three-month old normal mice with hematopoietic stem cells from control or oc/oc mice, which have defective acid secretion, and followed them for 12 to 28 weeks.Engraftment levels were assessed by flow cytometry of peripheral blood. Serum samples were collected every six weeks for measurement of bone turnover markers. At termination bones were collected for µCT and mechanical testing. An engraftment level of 98% was obtained. From week 6 until termination bone resorption was significantly reduced, while the osteoclast number was increased when comparing oc/oc to controls. Bone formation was elevated at week 6, normalized at week 12, and reduced onwards. µCT and mechanical analyses of femurs and vertebrae showed increased bone volume and bone strength of cortical and trabecular bone.In conclusion, these data show that attenuation of acid secretion in adult mice leads to uncoupling and improves bone strength.
Assuntos
Densidade Óssea , Reabsorção Óssea/fisiopatologia , Osteoclastos/enzimologia , Osteogênese/fisiologia , ATPases Vacuolares Próton-Translocadoras/fisiologia , Animais , Sobrevivência de Enxerto , Transplante de Células-Tronco Hematopoéticas , Camundongos , Osteopetrose/etiologia , Osteopetrose/prevenção & controleRESUMO
Infantile malignant osteopetrosis (IMO) is caused by lack of functional osteoclasts leading to skeletal abnormalities, blindness owing to compression of the optic nerves, bone marrow (BM) failure, and early death. In most patients, TCIRG1, a proton pump subunit essential for bone resorption, is mutated. oc/oc mice represent a model for IMO owing to a deletion in Tcirg1 and die around 4 weeks of age. To determine if hematopoietic stem cell transplantation without prior conditioning can reverse osteopetrosis, neonatal mice were transplanted intravenously with lineage-depleted BM cells. More than 85% of oc/oc mice transplanted with 5 × 10(6) cells survived long term with an engraftment of 3% to 5% in peripheral blood (PB). At 3 weeks, engraftment in the BM was 1% to 2%, but the cellularity had increased 60-fold compared with untreated oc/oc mice, and RANKL and macrophage colony-stimulating factor (M-CSF) expression in the BM was normalized. Histopathology and micro-computed tomography revealed almost complete reversal of osteopetrosis after 4 weeks. In vitro studies showed that bone resorption by osteoclasts from transplanted oc/oc mice was 14% of transplanted controls, and immunofluorescence microscopy revealed that resorption was mainly associated with osteoclasts of donor origin. Lineage analysis of BM, PB, and spleen did not provide any evidence for selective recruitment of cells to the osteoclastic lineage. The vision also was preserved in transplanted oc/oc mice, as determined by a visual tracking drum test. In summary, nonablative neonatal transplantation leading to engraftment of only a small fraction of normal cells rapidly reverses severe osteopetrosis in the oc/oc mouse model.
Assuntos
Transplante de Medula Óssea , Osteoclastos/citologia , Animais , Animais Recém-Nascidos , Camundongos , Camundongos Endogâmicos C3H , Tomografia Computadorizada por Raios XRESUMO
The recently revitalized interest in the regulation of hematopoietic stem cells (HSCs) by the bone marrow microenvironment has resulted in the identification of some important cell types that potentially form the HSC niche. The term 'osteoblast' has commonly been used to describe the endosteal elements of the HSC niche, but these cells are part of a larger family that functions in bone at different stages of differentiation. Given that there is much controversy as to what cell types have important roles in the HSC niche, this review offers an overview of the diverse osteoblastic cell types and discusses the current evidence regarding what roles they have in the HSC niche.
Assuntos
Células-Tronco Hematopoéticas/fisiologia , Osteoblastos/fisiologia , Nicho de Células-Tronco/fisiologia , Animais , Medula Óssea/fisiologia , Células da Medula Óssea/fisiologia , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/deficiência , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/fisiologia , Osso e Ossos/anatomia & histologia , Osso e Ossos/citologia , Linhagem da Célula , Humanos , Células-Tronco Mesenquimais/fisiologia , Receptor Tipo 1 de Hormônio Paratireóideo/fisiologia , Terminologia como AssuntoRESUMO
OBJECTIVE: Infantile malignant osteopetrosis is a fatal disease caused by lack of functional osteoclasts. In most of patients, TCIRG1, encoding a subunit of a proton pump essential for bone resorption, is mutated. Osteopetrosis leads to bone marrow failure and blindness due to optic nerve compression. Oc/oc mice have a deletion in Tcirg1 and die around 3 to 4 weeks, but can be rescued by neonatal stem cell transplantation (SCT) after irradiation conditioning. However, as irradiation of neonatal mice results in retinal degeneration, we wanted to investigate whether conditioning with busulphan prior to SCT can lead to preservation of vision and reversal of osteopetrosis in the oc/oc mouse model. MATERIALS AND METHODS: Pregnant dams were conditioned with busulphan and their litters transplanted with 1 x 10(6) normal lineage-depleted bone marrow cells intravenously or intraperitoneally. Mice were followed in terms of survival and engraftment level, as well as with peripheral blood lineage analysis, bone and eye histopathology and a visual-tracking drum test to assess vision. RESULTS: Busulphan at 15 mg/kg was toxic to oc/oc mice. However, six of seven oc/oc mice conditioned with busulphan 7.5 mg/kg survived past the normal lifespan with 10% engraftment, correction of the skeletal phenotype, and normalization of peripheral blood lineages. Busulphan, in contrast to irradiation, did not have adverse effects on the retina as determined by histopathology, and 8 weeks after transplantation control and oc/oc mice retained their vision. CONCLUSION: Low-dose busulphan conditioning and neonatal SCT leads to prolonged survival of oc/oc mice, reverses osteopetrosis and prevents blindness even at low engraftment levels.