RESUMO
Regulatory myeloid immune cells, such as myeloid-derived suppressor cells (MDSCs), populate inflamed or cancerous tissue and block immune cell effector functions. The lack of mechanistic insight into MDSC suppressive activity and a marker for their identification has hampered attempts to overcome T cell inhibition and unleash anti-cancer immunity. Here, we report that human MDSCs were characterized by strongly reduced metabolism and conferred this compromised metabolic state to CD8+ T cells, thereby paralyzing their effector functions. We identified accumulation of the dicarbonyl radical methylglyoxal, generated by semicarbazide-sensitive amine oxidase, to cause the metabolic phenotype of MDSCs and MDSC-mediated paralysis of CD8+ T cells. In a murine cancer model, neutralization of dicarbonyl activity overcame MDSC-mediated T cell suppression and, together with checkpoint inhibition, improved the efficacy of cancer immune therapy. Our results identify the dicarbonyl methylglyoxal as a marker metabolite for MDSCs that mediates T cell paralysis and can serve as a target to improve cancer immune therapy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Imunoterapia/métodos , Melanoma/imunologia , Células Supressoras Mieloides/imunologia , Aldeído Pirúvico/metabolismo , Amina Oxidase (contendo Cobre)/metabolismo , Animais , Linfócitos T CD8-Positivos/transplante , Comunicação Celular , Proliferação de Células , Humanos , Tolerância Imunológica , Ativação Linfocitária , Melanoma Experimental , Camundongos , Camundongos Transgênicos , Neoplasias Experimentais , Receptor de Morte Celular Programada 1/metabolismoRESUMO
Diagnostics for suspected mitochondrial disease (MD) can be challenging and necessitate invasive procedures like muscle biopsy. This is due to the extremely broad genetic and phenotypic spectrum, disease genes on both nuclear and mitochondrial DNA (mtDNA), and the tissue specificity of mtDNA variants. Exome sequencing (ES) has revolutionized the diagnostics for MD. However, the nuclear and mtDNA are investigated with separate tests, increasing costs and duration of diagnostics. The full potential of ES is often not exploited as the additional analysis of "off-target reads" deriving from the mtDNA can be used to analyze both genomes. We performed mtDNA analysis by ES of 2111 cases in a clinical setting. We further assessed the recall rate and precision as well as the estimation of heteroplasmy by ES data by comparison with targeted mtDNA next generation sequencing in 49 cases. ES identified known pathogenic mtDNA point mutations in 38 individuals, increasing the diagnostic yield by nearly 2%. Analysis of mtDNA variants by ES had a high recall rate (96.2 ± 5.6%) and an excellent precision (99.5 ± 2.2%) when compared to the gold standard of targeted mtDNA next generation sequencing. ES estimated heteroplasmy levels with an average difference of 6.6 ± 3.8%, sufficient for clinical decision making. Taken together, the mtDNA analysis from ES is of sufficient quality for clinical diagnostics. We therefore propose ES, investigating both nuclear and mtDNA, as first line test in individuals with suspected MD. One should be aware, that a negative result does not exclude MD and necessitates further test (in additional tissues).
Assuntos
Núcleo Celular/genética , Análise Mutacional de DNA/métodos , DNA Mitocondrial/genética , Exoma/genética , Doenças Mitocondriais/diagnóstico , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Masculino , Doenças Mitocondriais/genética , Adulto JovemRESUMO
OBJECTIVES: Isolated complex IV (cytochrome c oxidase) deficiency is one of the most frequent respiratory chain defects in mitochondrial disorders (MIDs) and usually occurs together with severe pediatric or rarely adult multisystem disease. Here we report an adult with isolated complex IV deficiency with unusually mild clinical manifestations. CASE REPORT: A 50-year-old man had developed generalized muscle aches and occasional twitching and stiffness of the musculature since age 48 years. He had a previous history of diabetes, acute hearing loss, hyperlipidemia, hyperuricemia, arterial hypertension, polyarthrosis, hypogonadism, and hypothyroidism. The family history was positive for diabetes (mother), CK elevation (brother), myalgias (brother), and proximal weakness of the upper limbs (mother). Work-up revealed hypoacusis, postural tremor and reduced tendon reflexes, recurrent mild hyper-CK-emia, neurogenic needle electromyography, and a muscle biopsy with mild non-specific changes. Biochemical investigations of the muscle homogenate revealed an isolated complex IV defect and reduced amounts of coenzyme Q (CoQ). He profited from CoQ supplementation, low-carbohydrate diet, and gluten-free diet. CONCLUSIONS: Isolated complex IV deficiency may present with only mild muscular, endocrine, or cardiac manifestations in adults. Coenzyme Q supplementation, low-carbohydrate diet, and gluten-free diet may have a beneficial effect at least on some of the manifestations.
Assuntos
Deficiência de Citocromo-c Oxidase/complicações , Deficiência de Citocromo-c Oxidase/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , FenótipoRESUMO
Mitochondrial DNA depletion syndrome (MDS) is usually a severe disorder of infancy or childhood, due to a reduced copy number of mtDNA molecules. MDS with only mild, nonspecific clinical manifestations and onset in adulthood has not been reported. A 47-year-old Caucasian female with short stature and a history of migraine, endometriosis, Crohn's disease, C-cell carcinoma of the thyroid gland, and a family history positive for mitochondrial disorder (2 sisters, aunt, niece), developed day-time sleepiness, exercise intolerance, and myalgias in the lower-limb muscles since age 46y. She slept 9-10 hours during the night and 2 hours after lunch daily. Clinical exam revealed sore neck muscles, bilateral ptosis, and reduced Achilles tendon reflexes exclusively. Blood tests revealed hyperlipidemia exclusively. Nerve conduction studies, needle electromyography, and cerebral and spinal magnetic resonance imaging were noninformative. Muscle biopsy revealed detached lobulated fibers with subsarcolemmal accentuation of the NADH and SDH staining. Realtime polymerase chain reaction revealed depletion of the mtDNA down to 9% of normal. MDS may be associated with a mild phenotype in adults and may not significantly progress during the first year after onset. In an adult with hypersomnia, severe tiredness, exercise intolerance, and a family history positive for mitochondrial disorder, a MDS should be considered.
RESUMO
Myoclonic epilepsy with ragged red fibres (MERRF) and mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) are established phenotypes of mitochondrial encephalopathies. Nearly all patients affected by MERRF harbour a mutation in the mitochondrial tRNA(Lys) gene. We report a 13-year-old patient who presented with the classical phenotype of MERRF but was found with the typical mutation of MELAS. The patient presented with myoclonic epilepsy beginning at 10 years of age, a muscle biopsy with ragged red fibres and some COX negative fibres and progressive bilateral MRI hyperintensitivities in the basal ganglia constituting MERRF syndrome but lacked clinical characteristics of MELAS. In particular, stroke-like episodes or lactic acidosis were not present. None of the tRNA mutations described in MERRF were found. However, further analyses showed the tRNA(Leu) mutation m.3243A>G usually found in MELAS to be responsible for the condition in this patient. This report highlights the broad phenotypic variability of mitochondrial encephalopathies with juvenile onset. It shows that m.3243A>G mutations can cause classical MERRF and emphasises the significance of comprehensive genetic studies if mitochondrial disease is suspected clinically.
Assuntos
Síndrome MERRF/genética , Músculos/patologia , RNA de Transferência de Leucina/genética , RNA/genética , Adolescente , DNA Mitocondrial , Humanos , Síndrome MELAS/genética , Imageamento por Ressonância Magnética , Mutação , Fenótipo , RNA Mitocondrial , ConvulsõesRESUMO
The Tim23 protein is the key component of the mitochondrial import machinery. It locates to the inner mitochondrial membrane and its own import is dependent on the DDP1/TIM13 complex. Mutations in human DDP1 cause the Mohr-Tranebjaerg syndrome (MTS/DFN-1; OMIM #304700), which is one of the two known human diseases of the mitochondrial protein import machinery. We created a Tim23 knockout mouse from a gene trap embryonic stem cell clone. Homozygous Tim23 mice were not viable. Heterozygous F1 mutants showed a 50% reduction of Tim23 protein in Western blot, a neurological phenotype and a markedly reduced life span. Haploinsufficiency of the Tim23 mutation underlines the critical role of the mitochondrial import machinery for maintaining mitochondrial function.
Assuntos
Expectativa de Vida , Proteínas de Membrana/deficiência , Mitocôndrias/genética , Mutação , Animais , Blastocisto/fisiologia , Membro Anterior/fisiologia , Genótipo , Força da Mão , Humanos , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Knockout , Proteínas de Transporte da Membrana Mitocondrial , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/deficiência , Proteínas Mitocondriais/genética , Síndromes Orofaciodigitais/genética , Fenótipo , Transporte Proteico/genética , Teste de Desempenho do Rota-RodRESUMO
Precursor proteins of the solute carrier family and of channel forming Tim components are imported into mitochondria in two main steps. First, they are translocated through the TOM complex in the outer membrane, a process assisted by the Tim9/Tim10 complex. They are passed on to the TIM22 complex, which facilitates their insertion into the inner membrane. In the present study, we have analyzed the function of the Tim9/Tim10 complex in the translocation of substrates across the outer membrane of mitochondria. The purified TOM core complex was reconstituted into lipid vesicles in which purified Tim9/Tim10 complex was entrapped. The precursor of the ADP/ATP carrier (AAC) was found to be translocated across the membrane of such lipid vesicles. Thus, these components are sufficient for translocation of AAC precursor across the outer membrane. Peptide libraries covering various substrate proteins were used to identify segments that are bound by Tim9/Tim10 complex upon translocation through the TOM complex. The patterns of binding sites on the substrate proteins suggest a mechanism by which portions of membrane-spanning segments together with flanking hydrophilic segments are recognized and bound by the Tim9/Tim10 complex as they emerge from the TOM complex into the intermembrane space.