RESUMO
AIMS: Resident and peripherally derived glioma associated microglia/macrophages (GAMM) play a key role in driving tumour progression, angiogenesis, invasion and attenuating host immune responses. Differentiating these cells' origins is challenging and current preclinical models such as irradiation-based adoptive transfer, parabiosis and transgenic mice have limitations. We aimed to develop a novel nonmyeloablative transplantation (NMT) mouse model that permits high levels of peripheral chimerism without blood-brain barrier (BBB) damage or brain infiltration prior to tumour implantation. METHODS: NMT dosing was determined in C57BL/6J or Pep3/CD45.1 mice conditioned with concentrations of busulfan ranging from 25 mg/kg to 125 mg/kg. Donor haematopoietic cells labelled with eGFP or CD45.2 were injected via tail vein. Donor chimerism was measured in peripheral blood, bone marrow and spleen using flow cytometry. BBB integrity was assessed with anti-IgG and anti-fibrinogen antibodies. Immunocompetent chimerised animals were orthotopically implanted with murine glioma GL-261 cells. Central and peripheral cell contributions were assessed using immunohistochemistry and flow cytometry. GAMM subpopulation analysis of peripheral cells was performed using Ly6C/MHCII/MerTK/CD64. RESULTS: NMT achieves >80% haematopoietic chimerism by 12 weeks without BBB damage and normal life span. Bone marrow derived cells (BMDC) and peripheral macrophages accounted for approximately 45% of the GAMM population in GL-261 implanted tumours. Existing markers such as CD45 high/low proved inaccurate to determine central and peripheral populations while Ly6C/MHCII/MerTK/CD64 reliably differentiated GAMM subpopulations in chimerised and unchimerised mice. CONCLUSION: NMT is a powerful method for dissecting tumour microglia and macrophage subpopulations and can guide further investigation of BMDC subsets in glioma and neuro-inflammatory diseases.
Assuntos
Neoplasias Encefálicas/patologia , Glioma/patologia , Macrófagos/patologia , Microglia/patologia , Animais , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
In the light of clinical experience in infantile onset Pompe patients, the immunological impact on the tolerability and long-term efficacy of enzyme replacement therapy (ERT) for lysosomal storage disorders has come under renewed scrutiny. This article details the currently proposed immunological mechanisms involved in the development of anti-drug antibodies and the current therapies used in their treatment. Given the current understanding of the adaptive immune response, it focuses particularly on T cell dependent mechanisms and the paradigm of using lymphocytic negative selection as a predictor of antibody formation. This concept originally postulated in the 1970s, stipulated that the genotypically determined lack of production or production of a variant protein determines an individual's lymphocytic repertoire. This in turn is the key factor in determining the potential severity of an individual's immunological response to ERT. It also highlights the need for immunological assay standardization particularly those looking at describing the degree of functional impact, robust biochemical or clinical endpoints and detailed patient subgroup identification if the true evaluations of impact are to be realised.
Assuntos
Terapia de Reposição de Enzimas , Sistema Imunitário/fisiologia , Doenças por Armazenamento dos Lisossomos/tratamento farmacológico , Doenças por Armazenamento dos Lisossomos/imunologia , Formação de Anticorpos , Terapia de Reposição de Enzimas/efeitos adversos , Terapia Enzimática , Enzimas/imunologia , Humanos , Resultado do Tratamento , alfa-Glucosidases/imunologia , alfa-Glucosidases/uso terapêuticoRESUMO
In vivo T-cell depletion, using alemtuzumab therapy prior to SCT, can reduce the incidence of GVHD. This treatment has a potential to delay immune reconstitution resulting in increased morbidity due to viral illnesses. We retrospectively analyzed data on all pediatric patients with non-malignant disorders who received alemtuzumab-based conditioning regimens in our center over the last 10 yr (n = 91). Our data show an OS of 91.2%. The incidence of acute (grade 2-4) GVHD was 18.7% and that of chronic GVHD 5.5%. Viremia due to adenovirus, EBV and CMV was seen in 19.8%, 64.8% and 39.6% patients, respectively, with only two deaths attributed to viral infection (adenovirus). Chimerism level at three month was predictive of graft outcome. Nine patients, who had graft failure after first SCT, were salvaged with a second SCT using RIC and same donor (if available). Based on these results, we conclude that the use of in vivo T-cell depletion is safe, achieves good chimerism and does not lead to increased morbidity and mortality due to viral infections. It is associated with a reduced incidence of chronic GVHD.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Transplante de Células-Tronco Hematopoéticas/métodos , Linfócitos T/imunologia , Adenoviridae/metabolismo , Adolescente , Alemtuzumab , Anemia Aplástica/terapia , Criança , Pré-Escolar , Feminino , Doença Enxerto-Hospedeiro/etiologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Imunossupressores/uso terapêutico , Incidência , Lactente , Masculino , Doenças Metabólicas/terapia , Estudos Retrospectivos , Quimeras de Transplante , Condicionamento Pré-Transplante , Transplante Homólogo , Resultado do Tratamento , Doadores não Relacionados , Viremia/fisiopatologia , Adulto JovemRESUMO
Mucopolysaccharidosis IIIB (MPS IIIB) is a lysosomal storage disorder characterized by severe behavioural disturbances and progressive loss of cognitive and motor function. There is no effective treatment, but behavioural testing is a valuable tool to assess neurodegeneration and the effect of novel therapies in mouse models of disease. Several groups have evaluated behaviour in this model, but the data are inconsistent, often conflicting with patient natural history. We hypothesize that this discrepancy could be due to differences in open field habituation and home cage behaviour. Eight-month-old wild-type and MPS IIIB mice were tested in a 1-h open field test, performed 1.5 h after lights on, and a 24-h home cage behaviour test performed after 24 h of acclimatization. In the 1-h test, MPS IIIB mice were hyperactive, with increased rapid exploratory behaviour and reduced immobility time. No differences in anxiety were seen. Over the course of the test, differences became more pronounced with maximal effects at 1 h. The 24-hour home cage test was less reliable. There was evidence of increased hyperactivity in MPS IIIB mice, however, immobility was also increased, suggesting a level of inconsistency in this test. Performance of open field analysis within 1-2 h after lights on is probably critical to achieving maximal success as MPS IIIB mice have a peak in activity around this time. The open field test effectively identifies hyperactive behaviour in MPS IIIB mice and is a significant tool for evaluating effects of therapy on neurodegeneration.
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Acetilglucosaminidase/genética , Hipercinese/genética , Atividade Motora/genética , Mucopolissacaridose III/genética , Animais , Ansiedade/genética , Modelos Animais de Doenças , Meio Ambiente , Comportamento Exploratório/fisiologia , Camundongos , Camundongos KnockoutRESUMO
Immune responses against an introduced transgenic protein are a potential risk in many gene replacement strategies to treat genetic disease. We have developed a gene delivery approach for hemophilia B based on lentiviral expression of human factor IX in purified hematopoietic stem cells. In both normal C57Bl/6J and hemophilic 129/Sv recipient mice, we observed the production of therapeutic levels of human factor IX, persisting for at least a year with tolerance to human factor IX antigen. Secondary and tertiary recipients also demonstrate long-term production of therapeutic levels of human factor IX and tolerance, even at very low levels of donor chimerism. Furthermore, in hemophilic mice, partial functional correction of treated mice and phenotypic rescue is achieved. These data show the potential of a stem cell approach to gene delivery to tolerize recipients to a secreted foreign transgenic protein and, with appropriate modification, may be of use in developing treatments for other genetic disorders.
Assuntos
Fator IX/genética , Terapia Genética/métodos , HIV-1/genética , Hemofilia B/terapia , Transplante de Células-Tronco/métodos , Animais , Antígenos/imunologia , Células Cultivadas , Fator IX/metabolismo , Expressão Gênica , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/terapia , Genótipo , Proteínas de Fluorescência Verde/genética , Hemofilia B/sangue , Humanos , Tolerância Imunológica , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo , Células-Tronco/metabolismo , Células-Tronco/virologia , Fatores de Tempo , Transdução Genética/métodos , TransgenesRESUMO
Gene therapy for Duchenne muscular dystrophy has so far not been successful because of the difficulty in achieving efficient and permanent gene transfer to the large number of affected muscles and the development of immune reactions against vector and transgenic protein. In addition, the prenatal onset of disease complicates postnatal gene therapy. We have therefore proposed a fetal approach to overcome these barriers. We have applied beta-galactosidase expressing equine infectious anaemia virus (EIAV) lentiviruses pseudotyped with VSV-G by single or combined injection via different routes to the MF1 mouse fetus on day 15 of gestation and describe substantial gene delivery to the musculature. Highly efficient gene transfer to skeletal muscles, including the diaphragm and intercostal muscles, as well as to cardiac myocytes was observed and gene expression persisted for at least 15 months after administration of this integrating vector. These findings support the concept of in utero gene delivery for therapeutic and long-term prevention/correction of muscular dystrophies and pave the way for a future application in the clinic.
Assuntos
Feto/metabolismo , Terapia Genética/métodos , Vírus da Anemia Infecciosa Equina/genética , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/terapia , beta-Galactosidase/genética , Animais , Feminino , Feto/imunologia , Expressão Gênica , Engenharia Genética , Injeções , Camundongos , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/embriologia , GravidezRESUMO
The liver is normally proliferatively quiescent, but hepatocyte loss through partial hepatectomy, uncomplicated by virus infection or inflammation, invokes a rapid regenerative response from all cell types in the liver to perfectly restore liver mass. Moreover, hepatocyte transplants in animals have shown that a certain proportion of hepatocytes in foetal and adult liver can clonally expand, suggesting that hepatoblasts/hepatocytes are themselves the functional stem cells of the liver. More severe liver injury can activate a potential stem cell compartment located within the intrahepatic biliary tree, giving rise to cords of bipotential transit amplifying cells (oval cells), that can ultimately differentiate into hepatocytes and biliary epithelial cells. A third population of stem cells with hepatic potential resides in the bone marrow; these haematopoietic stem cells may contribute to the albeit low renewal rate of hepatocytes, but can make a more significant contribution to regeneration under a very strong positive selection pressure. In such instances, cell fusion rather than transdifferentiation appears to be the underlying mechanism by which the haematopoietic genome becomes reprogrammed.
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Fígado/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular , Células-Tronco Hematopoéticas/citologia , HumanosRESUMO
The presence of CpG motifs and their associated sequences in bacterial DNA causes an immunotoxic response following the delivery of these plasmid vectors into mammalian hosts. We describe a biotechnological approach to the elimination of this problem by the creation of a bacterial cre recombinase expression system, tightly controlled by the arabinose regulon. This permits the Cre-mediated and -directed excision of the entire bacterial vector sequences from plasmid constructs to create supercoiled gene expression minicircles for gene therapy. Minicircle yields using standard culture volumes are sufficient for most in vitro and in vivo applications whereas minicircle expression in vitro is significantly increased over standard plasmid transfection. By the simple expedient of removing the bacterial DNA complement, we significantly reduce the size and CpG content of these expression vectors, which should also reduce DNA-induced inflammatory responses in a dose-dependent manner. We further describe the generation of minicircle expression vectors for mammalian mitochondrial gene therapy, for which no other vector systems currently exist. The removal of bacterial vector sequences should permit appropriate transcription and correct transcriptional cleavage from the mitochondrial minicircle constructs in a mitochondrial environment and brings the realization of mitochondrial gene therapy a step closer.