RESUMEN
The DNA hypomethylating agents (HMA) azacitidine (AZA) and decitabine (DAC) improve survival and transfusion independence in myelodysplastic syndrome (MDS) and enable a low intensity cytotoxic treatment for aged AML patients unsuitable for intensive chemotherapy, particularly in combination with novel agents. The proposed mechanism of AZA and DAC relies on active DNA replication and therefore patient responses are only observed after multiple cycles of treatment. Although extended dosing may provide the optimal scheduling, the reliance of injectable formulation of the drug limits it to intermittent treatment. Recently, an oral formulation of AZA demonstrated significantly improved patient relapse free survival (RFS) and overall survival (OS) when used as maintenance after chemotherapy for AML. In addition, both DAC and AZA were found to be highly effective to improve survival in elderly patients with AML through combination with other drugs. These recent exciting results have changed the therapeutic paradigm for elderly patients with AML. In light of this, we review current knowledge on HMA mechanism of action, clinical trials exploring dosing and scheduling, and recent HMA combination therapies to enhance efficacy.
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Leucemia Mieloide Aguda , Síndromes Mielodisplásicos , Anciano , Azacitidina/uso terapéutico , Decitabina/uso terapéutico , Terapia Genética , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Síndromes Mielodisplásicos/tratamiento farmacológico , Síndromes Mielodisplásicos/genética , Resultado del TratamientoRESUMEN
Myeloproliferative neoplasms (MPNs) are a group of blood cancers that arise following the sequential acquisition of genetic lesions in hematopoietic stem and progenitor cells (HSPCs). We identify mutational cooperation between Jak2V617F expression and Dnmt3a loss that drives progression from early-stage polycythemia vera to advanced myelofibrosis. Using in vivo, clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated protein 9 (Cas9) disruption of Dnmt3a in Jak2V617F knockin HSPC, we show that Dnmt3a loss blocks the accumulation of erythroid elements and causes fibrotic infiltration within the bone marrow and spleen. Transcriptional analysis and integration with human data sets identified a core DNMT3A-driven gene-expression program shared across multiple models and contexts of Dnmt3a loss. Aberrant self-renewal and inflammatory signaling were seen in Dnmt3a-/- Jak2V617F HSPC, driven by increased chromatin accessibility at enhancer elements. These findings identify oncogenic cooperativity between Jak2V617F-driven MPN and Dnmt3a loss, leading to activation of HSPC enhancer-driven inflammatory signaling.
Asunto(s)
Sustitución de Aminoácidos , ADN (Citosina-5-)-Metiltransferasas , Neoplasias Hematológicas , Células Madre Hematopoyéticas , Mutación Missense , Mielofibrosis Primaria , Transducción de Señal/genética , Animales , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN Metiltransferasa 3A , Neoplasias Hematológicas/enzimología , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patología , Células Madre Hematopoyéticas/enzimología , Células Madre Hematopoyéticas/patología , Humanos , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Ratones , Ratones Mutantes , Mielofibrosis Primaria/enzimología , Mielofibrosis Primaria/genética , Mielofibrosis Primaria/patologíaRESUMEN
Hematopoietic stem and progenitor cells (HSPCs) are vulnerable to endogenous damage and defects in DNA repair can limit their function. The 2 single-stranded DNA (ssDNA) binding proteins SSB1 and SSB2 are crucial regulators of the DNA damage response; however, their overlapping roles during normal physiology are incompletely understood. We generated mice in which both Ssb1 and Ssb2 were constitutively or conditionally deleted. Constitutive Ssb1/Ssb2 double knockout (DKO) caused early embryonic lethality, whereas conditional Ssb1/Ssb2 double knockout (cDKO) in adult mice resulted in acute lethality due to bone marrow failure and intestinal atrophy featuring stem and progenitor cell depletion, a phenotype unexpected from the previously reported single knockout models of Ssb1 or Ssb2 Mechanistically, cDKO HSPCs showed altered replication fork dynamics, massive accumulation of DNA damage, genome-wide double-strand breaks enriched at Ssb-binding regions and CpG islands, together with the accumulation of R-loops and cytosolic ssDNA. Transcriptional profiling of cDKO HSPCs revealed the activation of p53 and interferon (IFN) pathways, which enforced cell cycling in quiescent HSPCs, resulting in their apoptotic death. The rapid cell death phenotype was reproducible in in vitro cultured cDKO-hematopoietic stem cells, which were significantly rescued by nucleotide supplementation or after depletion of p53. Collectively, Ssb1 and Ssb2 control crucial aspects of HSPC function, including proliferation and survival in vivo by resolving replicative stress to maintain genomic stability.
Asunto(s)
Proliferación Celular/fisiología , Roturas del ADN de Doble Cadena , Inestabilidad Genómica/fisiología , Células Madre Hematopoyéticas/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Animales , Supervivencia Celular/fisiología , Islas de CpG/fisiología , Células Madre Hematopoyéticas/citología , Ratones , Ratones Noqueados , Proteínas Supresoras de la Señalización de Citocinas/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Granulocyte colony-stimulating factor (G-CSF) is widely used clinically to prevent neutropenia after cytotoxic chemotherapy and to mobilize hematopoietic stem cells (HSCs) for transplantation. Autophagy, a process of cytoplasmic component recycling, maintains cellular homeostasis and protects the cell during periods of metabolic stress or nutrient deprivation. We have observed that G-CSF activates autophagy in neutrophils and HSCs from both mouse and human donors. Furthermore, G-CSF-induced neutrophil and HSC mobilization is impaired in the absence of autophagy. In contrast, autophagy is dispensable for direct HSC mobilization in response to the CXCR4 antagonist AMD3100. Altogether, these data demonstrate an important role for G-CSF in invoking autophagy within hematopoietic and myeloid cells and suggest that this pathway is critical for ensuring cell survival in response to clinically relevant cytokine-induced stress. These findings have direct relevance to HSC transplantation and the increasing clinical use of agents that modulate autophagy.
Asunto(s)
Autofagia , Factor Estimulante de Colonias de Granulocitos/farmacología , Movilización de Célula Madre Hematopoyética , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/efectos de los fármacos , Animales , Fármacos Anti-VIH/farmacología , Antígenos CD34/genética , Antígenos CD34/metabolismo , Proteína 5 Relacionada con la Autofagia , Bencilaminas , Western Blotting , Células Cultivadas , Ciclamas , Citometría de Flujo , Células Madre Hematopoyéticas/patología , Compuestos Heterocíclicos/farmacología , Humanos , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/fisiología , Neutrófilos/efectos de los fármacos , Neutrófilos/patología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores CXCR4/antagonistas & inhibidores , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante AutólogoRESUMEN
SSB1 and SSB2 are newly identified single-stranded (ss) DNA binding proteins that play a crucial role in genome maintenance in humans. We recently generated a knockout mouse model of Ssb1 and revealed its essential role for neonatal survival. Notably, we found compensatory up-regulation of Ssb2 protein levels in multiple tissues of conditional Ssb1(-/-) mice, suggesting functional compensation between these 2 proteins. We report here the first description of Ssb2(-/-) knockout mice. Surprisingly, unlike Ssb1 knockout mice, Ssb2(-/-) mice are viable and fertile and do not exhibit marked phenotypic changes when compared with their Ssb2(+/+) and Ssb2(+/-) littermates. Notably, we did not detect any pathologic changes in the thymus, spleen, or testes, tissues with the most abundant expression of Ssb2. Moreover, Ssb2(-/-) mouse embryonic fibroblasts (MEFs) did not show any sensitivity to DNA-damaging agents, or defects in DNA repair capacity. However, we observed modest up-regulation of Ssb1 levels in Ssb2(-/-) MEFs as well as in Ssb2(-/-) thymus and spleen, suggesting that Ssb1 is likely able to compensate for the loss of Ssb2 in mice. Altogether, our results show that Ssb2 is dispensable for embryogenesis and adult tissue homeostasis, including thymopoiesis, splenic development, male fertility, and DNA repair in mice.
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Proteínas Portadoras/metabolismo , Reparación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Fertilidad/genética , Timo/metabolismo , Animales , Fibroblastos/metabolismo , Fibroblastos/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Unión Proteica/genética , Bazo/metabolismo , Bazo/fisiología , Timo/fisiología , Regulación hacia Arriba/genéticaRESUMEN
Effective erythropoiesis requires an appropriate supply of iron and mechanisms regulating iron homeostasis and erythropoiesis are intrinsically linked. Iron dysregulation, typified by iron-deficiency anaemia and iron overload, is common in many clinical conditions and impacts the health of up to 30% of the world's population. The proteins transmembrane protease, serine 6 (TMPRSS6; also termed matriptase-2), HFE and transferrin receptor 2 (TFR2) play important and opposing roles in systemic iron homeostasis, by regulating expression of the iron regulatory hormone hepcidin. We have performed a systematic analysis of mice deficient in these three proteins and show that TMPRSS6 predominates over HFE and TFR2 in hepcidin regulation. The phenotype of mice lacking TMPRSS6 and TFR2 is characterized by severe anaemia and extramedullary haematopoiesis in the spleen. Stress erythropoiesis in these mice results in increased expression of the newly identified erythroid iron regulator erythroferrone, which does not appear to overcome the hepcidin overproduction mediated by loss of TMPRSS6. Extended analysis reveals that TFR2 plays an important role in erythroid cells, where it is involved in terminal erythroblast differentiation and the regulation of erythropoietin. In conclusion, we have identified an essential role for TFR2 in erythropoiesis that may provide new targets for the treatment of anaemia.
Asunto(s)
Anemia Ferropénica/sangre , Eritropoyesis/fisiología , Receptores de Transferrina/fisiología , Anemia Ferropénica/metabolismo , Animales , Diferenciación Celular/fisiología , Células Eritroides/patología , Eritropoyetina/biosíntesis , Hematopoyesis Extramedular/fisiología , Proteína de la Hemocromatosis , Hepcidinas/metabolismo , Antígenos de Histocompatibilidad Clase I/sangre , Antígenos de Histocompatibilidad Clase I/fisiología , Riñón/metabolismo , Hígado/metabolismo , Masculino , Proteínas de la Membrana/sangre , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/fisiología , Ratones , Ratones Noqueados , Receptores de Eritropoyetina/metabolismo , Receptores de Transferrina/sangre , Receptores de Transferrina/deficiencia , Serina Endopeptidasas/sangre , Serina Endopeptidasas/deficiencia , Serina Endopeptidasas/fisiología , Esplenomegalia/sangreRESUMEN
Interferon-α (IFNα) is an effective treatment of patients with myeloproliferative neoplasms (MPNs). In addition to inducing hematological responses in most MPN patients, IFNα reduces the JAK2V617F allelic burden and can render the JAK2V617F mutant clone undetectable in some patients. The precise mechanism underlying these responses is incompletely understood and whether the molecular responses that are seen occur due to the effects of IFNα on JAK2V617F mutant stem cells is debated. Using a murine model of Jak2V617F MPN, we investigated the effects of IFNα on Jak2V617F MPN-propagating stem cells in vivo. We report that IFNα treatment induces hematological responses in the model and causes depletion of Jak2V617F MPN-propagating cells over time, impairing disease transplantation. We demonstrate that IFNα treatment induces cell cycle activation of Jak2V617F mutant long-term hematopoietic stem cells and promotes a predetermined erythroid-lineage differentiation program. These findings provide insights into the differential effects of IFNα on Jak2V617F mutant and normal hematopoiesis and suggest that IFNα achieves molecular remissions in MPN patients through its effects on MPN stem cells. Furthermore, these results support combinatorial therapeutic approaches in MPN by concurrently depleting dormant JAK2V617F MPN-propagating stem cells with IFNα and targeting the proliferating downstream progeny with JAK2 inhibitors or cytotoxic chemotherapy.
Asunto(s)
Neoplasias Hematológicas/patología , Interferón-alfa/farmacología , Janus Quinasa 2/genética , Células Madre Neoplásicas/efectos de los fármacos , Policitemia Vera/patología , Sustitución de Aminoácidos/genética , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Neoplasias Hematológicas/genética , Humanos , Janus Quinasa 2/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Células Madre Neoplásicas/fisiología , Fenilalanina/genética , Policitemia Vera/tratamiento farmacológico , Policitemia Vera/genética , Valina/genéticaRESUMEN
Streptococcus pyogenes ranks among the main causes of mortality from bacterial infections worldwide. Currently there is no vaccine to prevent diseases such as rheumatic heart disease and invasive streptococcal infection. The streptococcal M protein that is used as the substrate for epidemiological typing is both a virulence factor and a vaccine antigen. Over 220 variants of this protein have been described, making comparisons between proteins difficult, and hindering M protein-based vaccine development. A functional classification based on 48 emm-clusters containing closely related M proteins that share binding and structural properties is proposed. The need for a paradigm shift from type-specific immunity against S. pyogenes to emm-cluster based immunity for this bacterium should be further investigated. Implementation of this emm-cluster-based system as a standard typing scheme for S. pyogenes will facilitate the design of future studies of M protein function, streptococcal virulence, epidemiological surveillance, and vaccine development.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Vacunas Estreptocócicas/inmunología , Streptococcus pyogenes/clasificación , Streptococcus pyogenes/fisiología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Clonación Molecular , Datos de Secuencia Molecular , Filogenia , Proteínas RecombinantesRESUMEN
Murine models offer a valuable tool to recapitulate genetically defined subtypes of AML, and to assess the potential of compound mutations and clonal evolution during disease progression. This is of particular importance for difficult to treat leukemias such as FLT3 internal tandem duplication (ITD) positive AML. While conditional gene targeting by Cre recombinase is a powerful technology that has revolutionized biomedical research, consequences of Cre expression such as lack of fidelity, toxicity or off-target effects need to be taken into consideration. We report on a transgenic murine model of FLT3-ITD induced disease, where Cre recombinase expression alone, and in the absence of a conditional allele, gives rise to an aggressive leukemia phenotype. Here, expression of various Cre recombinases leads to polyclonal expansion of FLT3ITD/ITD progenitor cells, induction of a differentiation block and activation of Myc-dependent gene expression programs. Our report is intended to alert the scientific community of potential risks associated with using this specific mouse model and of unexpected effects of Cre expression when investigating cooperative oncogenic mutations in murine models of cancer.
Asunto(s)
Leucemia Mieloide Aguda , Animales , Ratones , Modelos Animales de Enfermedad , Tirosina Quinasa 3 Similar a fms/genética , Duplicación de Gen , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Ratones Transgénicos , MutaciónAsunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias Hematológicas/inmunología , Interleucina-3/inmunología , Janus Quinasa 2/inmunología , Trastornos Mieloproliferativos/inmunología , Receptores de Interleucina-3/inmunología , Animales , Médula Ósea/efectos de los fármacos , Médula Ósea/inmunología , Médula Ósea/patología , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/patología , Humanos , Interleucina-3/genética , Janus Quinasa 2/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/patología , Receptores de Interleucina-3/genética , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/inmunología , Transducción de Señal , Trombopoyetina/farmacologíaRESUMEN
The caudal-related homeobox transcription factor CDX2 is expressed in leukemic cells but not during normal blood formation. Retroviral overexpression of Cdx2 induces AML in mice, however the developmental stage at which CDX2 exerts its effect is unknown. We developed a conditionally inducible Cdx2 mouse model to determine the effects of in vivo, inducible Cdx2 expression in hematopoietic stem and progenitor cells (HSPCs). Cdx2-transgenic mice develop myelodysplastic syndrome with progression to acute leukemia associated with acquisition of additional driver mutations. Cdx2-expressing HSPCs demonstrate enrichment of hematopoietic-specific enhancers associated with pro-differentiation transcription factors. Furthermore, treatment of Cdx2 AML with azacitidine decreases leukemic burden. Extended scheduling of low-dose azacitidine shows greater efficacy in comparison to intermittent higher-dose azacitidine, linked to more specific epigenetic modulation. Conditional Cdx2 expression in HSPCs is an inducible model of de novo leukemic transformation and can be used to optimize treatment in high-risk AML.
Asunto(s)
Factor de Transcripción CDX2/metabolismo , Células Madre Hematopoyéticas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Síndromes Mielodisplásicos/metabolismo , Animales , Factor de Transcripción CDX2/genética , Transformación Celular Neoplásica , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/fisiopatología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/fisiopatologíaRESUMEN
JAK2V617F is the most common mutation in patients with BCR-ABL negative myeloproliferative neoplasms (MPNs). The eradication of JAK2V617F hematopoietic stem cells (HSCs) is critical for achieving molecular remissions and cure. We investigate the distinct effects of two therapies, ruxolitinib (JAK1/2 inhibitor) and interferon-alpha (IFN-α), on the disease-initiating HSC population. Whereas ruxolitinib inhibits Stat5 activation in erythroid progenitor populations, it fails to inhibit this same pathway in HSCs. In contrast, IFN-α has direct effects on HSCs. Furthermore, STAT1 phosphorylation and pathway activation is greater after IFN-α stimulation in Jak2V617F murine HSCs with increased induction of reactive oxygen species, DNA damage and reduction in quiescence after chronic IFN-α treatment. Interestingly, ruxolitinib does not block IFN-α induced reactive oxygen species and DNA damage in Jak2V617F murine HSCs in vivo. This work provides a mechanistic rationale informing how pegylated IFN-α reduces JAK2V617F allelic burden in the clinical setting and may inform future clinical efforts to combine ruxolitinib with pegylated IFN-α in patients with MPN.
Asunto(s)
Células Madre Hematopoyéticas/efectos de los fármacos , Interferón-alfa/farmacología , Janus Quinasa 2/genética , Mutación , Trastornos Mieloproliferativos/tratamiento farmacológico , Pirazoles/farmacología , Factor de Transcripción STAT1/metabolismo , Animales , Antivirales/farmacología , Proliferación Celular , Células Cultivadas , Quimioterapia Combinada , Femenino , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/patología , Nitrilos , Pirimidinas , Factor de Transcripción STAT1/genéticaRESUMEN
The C-terminal region of the M-protein of Streptococcus pyogenes is a major target for vaccine development. The major feature is the C-repeat region, consisting of 35-42 amino acid repeat units that display high but not perfect identity. SV1 is a S. pyogenes vaccine candidate that incorporates five 14mer amino acid sequences (called J14i variants) from differing C-repeat units in a single recombinant construct. Here we show that the J14i variants chosen for inclusion in SV1 are the most common variants in a dataset of 176 unique M-proteins. Murine antibodies raised against SV1 were shown to bind to each of the J14i variants present in SV1, as well as variants not present in the vaccine. Antibodies raised to the individual J14i variants were also shown to bind to multiple but different combinations of J14i variants, supporting the underlying rationale for the design of SV1. A Lewis Rat Model of valvulitis was then used to assess the capacity of SV1 to induce deleterious immune response associated with rheumatic heart disease. In this model, both SV1 and the M5 positive control protein were immunogenic. Neither of these antibodies were cross-reactive with cardiac myosin or collagen. Splenic T cells from SV1/CFA and SV1/alum immunized rats did not proliferate in response to cardiac myosin or collagen. Subsequent histological examination of heart tissue showed that 4 of 5 mice from the M5/CFA group had valvulitis and inflammatory cell infiltration into valvular tissue, whereas mice immunised with SV1/CFA, SV1/alum showed no sign of valvulitis. These results suggest that SV1 is a safe vaccine candidate that will elicit antibodies that recognise the vast majority of circulating GAS M-types.
Asunto(s)
Anticuerpos Antibacterianos/biosíntesis , Antígenos Bacterianos/inmunología , Cardiopatía Reumática/prevención & control , Infecciones Estreptocócicas/prevención & control , Vacunas Estreptocócicas/administración & dosificación , Streptococcus pyogenes/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Compuestos de Alumbre/administración & dosificación , Animales , Antígenos Bacterianos/genética , Colágeno/genética , Colágeno/metabolismo , Femenino , Expresión Génica , Válvulas Cardíacas/efectos de los fármacos , Válvulas Cardíacas/inmunología , Válvulas Cardíacas/microbiología , Válvulas Cardíacas/patología , Ratones , Ratones Endogámicos BALB C , Miosinas/genética , Miosinas/metabolismo , Ratas , Ratas Endogámicas Lew , Secuencias Repetitivas de Aminoácido , Cardiopatía Reumática/inmunología , Cardiopatía Reumática/microbiología , Cardiopatía Reumática/patología , Bazo/efectos de los fármacos , Bazo/inmunología , Bazo/microbiología , Bazo/patología , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/microbiología , Infecciones Estreptocócicas/patología , Vacunas Estreptocócicas/biosíntesis , Vacunas Estreptocócicas/inmunología , Streptococcus pyogenes/efectos de los fármacos , Streptococcus pyogenes/genética , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/microbiología , Linfocitos T/patología , Vacunas SintéticasRESUMEN
Acute myeloid leukemia (AML) is an aggressive and lethal blood cancer maintained by rare populations of leukemia stem cells (LSCs). Selective targeting of LSCs is a promising approach for treating AML and preventing relapse following chemotherapy, and developing such therapeutic modalities is a key priority. Here, we show that targeting telomerase activity eradicates AML LSCs. Genetic deletion of the telomerase subunit Terc in a retroviral mouse AML model induces cell-cycle arrest and apoptosis of LSCs, and depletion of telomerase-deficient LSCs is partially rescued by p53 knockdown. Murine Terc(-/-) LSCs express a specific gene expression signature that can be identified in human AML patient cohorts and is positively correlated with patient survival following chemotherapy. In xenografts of primary human AML, genetic or pharmacological inhibition of telomerase targets LSCs, impairs leukemia progression, and delays relapse following chemotherapy. Altogether, these results establish telomerase inhibition as an effective strategy for eliminating AML LSCs.
Asunto(s)
Indoles/administración & dosificación , Leucemia Mieloide Aguda/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Niacinamida/análogos & derivados , Telomerasa/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Células Cultivadas , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Inactivación de Genes , Humanos , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos C57BL , Células Madre Neoplásicas/fisiología , Células Madre Neoplásicas/trasplante , Niacinamida/administración & dosificación , Oligonucleótidos , ARN Interferente Pequeño/genética , Recurrencia , Telomerasa/genética , Proteína p53 Supresora de Tumor/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A major challenge for Streptococcus pyogenes vaccine development is the identification of epitopes that confer protection from infection by multiple S. pyogenes M-types. Here we have identified and characterised the distribution of common variant sequences from individual repeat units of the C-repeat region (CRR) of M-proteins representing 77 different M-types. Three polyvalent fusion vaccine candidates (SV1, SV2 and SV3) incorporating the most common variants were subsequently expressed and purified, and demonstrated to be alpha-helical by Circular Dichroism (CD), a secondary conformational characteristic of the CRR in the M-protein. Antibodies raised against each of these constructs recognise M-proteins that vary in their CRR, and bind to the surface of multiple S. pyogenes isolates. Antibodies raised against SV1, containing five variant sequences, also kill heterologous S. pyogenes isolates in in vitro bactericidal assays. Further structural characterisation of this construct demonstrated the conformation of SV1 was stable at different pHs, and thermal unfolding of SV1 is a reversible process. Our findings demonstrate that linkage of multiple variant sequences into a single recombinant construct overcomes the need to embed the variant sequences in foreign helix promoting flanking sequences for conformational stability, and demonstrates the viability of the polyvalent candidates as global S. pyogenes vaccine candidates.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas Portadoras/inmunología , Epítopos/inmunología , Vacunas Estreptocócicas/administración & dosificación , Vacunas Estreptocócicas/inmunología , Streptococcus pyogenes/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Actividad Bactericida de la Sangre , Proteínas Portadoras/química , Proteínas Portadoras/genética , Dicroismo Circular , Secuencia Conservada , Epítopos/genética , Femenino , Ratones , Viabilidad Microbiana/efectos de los fármacos , Conformación Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Secuencias Repetitivas de Aminoácido/genética , Secuencias Repetitivas de Aminoácido/inmunología , Vacunas Estreptocócicas/genética , Streptococcus pyogenes/genética , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunologíaRESUMEN
Infection of the skin or throat by Streptococcus dysgalactiae subspecies equisimilis (SDSE) may result in a number of human diseases. To understand mechanisms that give rise to new genetic variants in this species, we used multi-locus sequence typing (MLST) to characterise relationships in the SDSE population from India, a country where streptococcal disease is endemic. The study revealed Indian SDSE isolates have sequence types (STs) predominantly different to those reported from other regions of the world. Emm-ST combinations in India are also largely unique. Split decomposition analysis, the presence of emm-types in unrelated clonal complexes, and analysis of phylogenetic trees based on concatenated sequences all reveal an extensive history of recombination within the population. The ratio of recombination to mutation (r/m) events (11:1) and per site r/m ratio (41:1) in this population is twice as high as reported for SDSE from non-endemic regions. Recombination involving the emm-gene is also more frequent than recombination involving housekeeping genes, consistent with diversification of M proteins offering selective advantages to the pathogen. Our data demonstrate that genetic recombination in endemic regions is more frequent than non-endemic regions, and gives rise to novel local SDSE variants, some of which may have increased fitness or pathogenic potential.