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1.
Am J Physiol Lung Cell Mol Physiol ; 322(3): L438-L448, 2022 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-35043685

RESUMEN

Hereditary pulmonary alveolar proteinosis (hPAP) is a rare disorder caused by recessive mutations in GM-CSF receptor subunit α/ß genes (CSF2RA/CSF2RB, respectively) characterized by impaired GM-CSF-dependent surfactant clearance by alveolar macrophages (AMs) resulting in alveolar surfactant accumulation and hypoxemic respiratory failure. Because hPAP is caused by CSF2RA mutations in most patients, we created an animal model of hPAP caused by Csf2ra gene disruption (Csf2ra-/- mice) and evaluated the effects on AMs and lungs. Macrophages from Csf2ra-/- mice were unable to bind and clear GM-CSF, did not exhibit GM-CSF signaling, and had functional defects in phagocytosis, cholesterol clearance, and surfactant clearance. Csf2ra-/- mice developed a time-dependent, progressive lung disease similar to hPAP in children caused by CSF2RA mutations with respect to the clinical, physiological, histopathological, biochemical abnormalities, biomarkers of PAP lung disease, and clinical course. In contrast, Csf2ra+/- mice had functionally normal AMs and no lung disease. Pulmonary macrophage transplantation (PMT) without myeloablation resulted in long-term engraftment, restoration of GM-CSF responsiveness to AMs, and a safe and durable treatment effect that lasted for the duration of the experiment (6 mo). Results demonstrate that homozygous (but not heterozygous) Csf2ra gene ablation caused hPAP identical to hPAP in children with CSF2RA mutations, identified AMs as the cellular site of hPAP pathogenesis in Csf2ra-/- mice, and have implications for preclinical studies supporting the translation of PMT as therapy of hPAP in humans.


Asunto(s)
Proteinosis Alveolar Pulmonar , Surfactantes Pulmonares , Animales , Modelos Animales de Enfermedad , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Humanos , Macrófagos Alveolares/metabolismo , Ratones , Proteinosis Alveolar Pulmonar/genética , Proteinosis Alveolar Pulmonar/metabolismo , Surfactantes Pulmonares/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Tensoactivos/metabolismo
2.
Mol Ther ; 27(9): 1597-1611, 2019 09 04.
Artículo en Inglés | MEDLINE | ID: mdl-31326401

RESUMEN

Hereditary pulmonary alveolar proteinosis (PAP) is a genetic lung disease characterized by surfactant accumulation and respiratory failure arising from disruption of GM-CSF signaling. While mutations in either CSF2RA or CSF2RB (encoding GM-CSF receptor α or ß chains, respectively) can cause PAP, α chain mutations are responsible in most patients. Pulmonary macrophage transplantation (PMT) is a promising new cell therapy in development; however, no studies have evaluated this approach for hereditary PAP (hPAP) caused by Csf2ra mutations. Here, we report on the preclinical safety, tolerability, and efficacy of lentiviral-vector (LV)-mediated Csf2ra expression in macrophages and PMT of gene-corrected macrophages (gene-PMT therapy) in Csf2ra gene-ablated (Csf2ra-/-) mice. Gene-PMT therapy resulted in a stable transgene integration and correction of GM-CSF signaling and functions in Csf2ra-/- macrophages in vitro and in vivo and resulted in engraftment and long-term persistence of gene-corrected macrophages in alveoli; restoration of pulmonary surfactant homeostasis; correction of PAP-specific cytologic, histologic, and biomarker abnormalities; and reduced inflammation associated with disease progression in untreated mice. No adverse consequences of gene-PMT therapy in Csf2ra-/- mice were observed. Results demonstrate that gene-PMT therapy of hPAP in Csf2ra-/- mice was highly efficacious, durable, safe, and well tolerated.


Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos , Terapia Genética , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/trasplante , Proteinosis Alveolar Pulmonar/genética , Proteinosis Alveolar Pulmonar/terapia , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Animales , Proliferación Celular , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Modelos Animales de Enfermedad , Expresión Génica , Terapia Genética/métodos , Vectores Genéticos/genética , Inmunofenotipificación , Lentivirus/genética , Ratones , Ratones Noqueados , Proteinosis Alveolar Pulmonar/diagnóstico , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Transducción de Señal , Transducción Genética
3.
Mol Ther Methods Clin Dev ; 32(2): 101213, 2024 Jun 13.
Artículo en Inglés | MEDLINE | ID: mdl-38596536

RESUMEN

Pulmonary macrophage transplantation (PMT) is a gene and cell transplantation approach in development as therapy for hereditary pulmonary alveolar proteinosis (hPAP), a surfactant accumulation disorder caused by mutations in CSF2RA/B (and murine homologs). We conducted a toxicology study of PMT of Csf2ra gene-corrected macrophages (mGM-Rα+Mϕs) or saline-control intervention in Csf2raKO or wild-type (WT) mice including single ascending dose and repeat ascending dose studies evaluating safety, tolerability, pharmacokinetics, and pharmacodynamics. Lentiviral-mediated Csf2ra cDNA transfer restored GM-CSF signaling in mGM-Rα+Mϕs. Following PMT, mGM-Rα+Mϕs engrafted, remained within the lungs, and did not undergo uncontrolled proliferation or result in bronchospasm, pulmonary function abnormalities, pulmonary or systemic inflammation, anti-transgene product antibodies, or pulmonary fibrosis. Aggressive male fighting caused a similarly low rate of serious adverse events in saline- and PMT-treated mice. Transient, minor pulmonary neutrophilia and exacerbation of pre-existing hPAP-related lymphocytosis were observed 14 days after PMT of the safety margin dose but not the target dose (5,000,000 or 500,000 mGM-Rα+Mϕs, respectively) and only in Csf2raKO mice but not in WT mice. PMT reduced lung disease severity in Csf2raKO mice. Results indicate PMT of mGM-Rα+Mϕs was safe, well tolerated, and therapeutically efficacious in Csf2raKO mice, and established a no adverse effect level and 10-fold safety margin.

4.
Mitochondrion ; 62: 85-101, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-34740864

RESUMEN

Granulocyte-macrophage colony-stimulating factor (GM-CSF) exerts pleiotropic effects on macrophages and is required for self-renewal but the mechanisms responsible are unknown. Using mouse models with disrupted GM-CSF signaling, we show GM-CSF is critical for mitochondrial turnover, functions, and integrity. GM-CSF signaling is essential for fatty acid ß-oxidation and markedly increased tricarboxylic acid cycle activity, oxidative phosphorylation, and ATP production. GM-CSF also regulated cytosolic pathways including glycolysis, pentose phosphate pathway, and amino acid synthesis. We conclude that GM-CSF regulates macrophages in part through a critical role in maintaining mitochondria, which are necessary for cellular metabolism as well as proliferation and self-renewal.


Asunto(s)
Proliferación Celular/fisiología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Macrófagos/fisiología , Mitocondrias/metabolismo , Animales , Células de la Médula Ósea , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Masculino , Ratones , Ratones Noqueados
5.
J Clin Invest ; 116(9): 2366-77, 2006 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16955139

RESUMEN

Many intracellular pathogens, including Toxoplasma gondii, survive within macrophages by residing in vacuoles that avoid fusion with lysosomes. It is important to determine whether cell-mediated immunity can trigger macrophage antimicrobial activity by rerouting these vacuoles to lysosomes. We report that CD40 stimulation of human and mouse macrophages infected with T. gondii resulted in fusion of parasitophorous vacuoles and late endosomes/lysosomes. Vacuole/lysosome fusion took place even when CD40 was ligated after the formation of parasitophorous vacuoles. Genetic and pharmacological approaches that impaired phosphoinositide-3-class 3 (PIK3C3), Rab7, vacuolar ATPase, and lysosomal enzymes revealed that vacuole/lysosome fusion mediated antimicrobial activity induced by CD40. Ligation of CD40 caused colocalization of parasitophorous vacuoles and LC3, a marker of autophagy, which is a process that controls lysosomal degradation. Vacuole/lysosome fusion and antimicrobial activity were shown to be dependent on autophagy. Thus, cell-mediated immunity through CD40 stimulation can reroute an intracellular pathogen to the lysosomal compartment, resulting in macrophage antimicrobial activity.


Asunto(s)
Antígenos CD40/inmunología , Lisosomas/fisiología , Activación de Macrófagos/inmunología , Macrófagos Peritoneales/inmunología , Macrófagos/inmunología , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Vacuolas/fisiología , Animales , Animales Modificados Genéticamente , Autofagia , Técnicas de Cultivo de Célula , Cloranfenicol O-Acetiltransferasa/genética , Humanos , Interferón gamma/deficiencia , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/fisiología , Linfocitos T/inmunología , Toxoplasma/genética
6.
Sci Rep ; 7(1): 10211, 2017 08 31.
Artículo en Inglés | MEDLINE | ID: mdl-28860566

RESUMEN

Macrophages are critical to organ structure and function in health and disease. To determine mechanisms by which granulocyte/macrophage-colony stimulating factor (GM-CSF) signaling normally maintains surfactant homeostasis and how its disruption causes pulmonary alveolar proteinosis (PAP), we evaluated lipid composition in alveolar macrophages and lung surfactant, macrophage-mediated surfactant clearance kinetics/dynamics, and cholesterol-targeted pharmacotherapy of PAP in vitro and in vivo. Without GM-CSF signaling, surfactant-exposed macrophages massively accumulated cholesterol ester-rich lipid-droplets and surfactant had an increased proportion of cholesterol. GM-CSF regulated cholesterol clearance in macrophages in constitutive, dose-dependent, and reversible fashion but did not affect phospholipid clearance. PPARγ-agonist therapy increased cholesterol clearance in macrophages and reduced disease severity in PAP mice. Results demonstrate that GM-CSF is required for cholesterol clearance in macrophages, identify reduced cholesterol clearance as the primary macrophage defect driving PAP pathogenesis, and support the feasibility of translating pioglitazone as a novel pharmacotherapy of PAP.


Asunto(s)
Colesterol/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Pioglitazona/administración & dosificación , Proteinosis Alveolar Pulmonar/tratamiento farmacológico , Animales , Diferenciación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Homeostasis/efectos de los fármacos , Humanos , Macrófagos Alveolares/citología , Macrófagos Alveolares/metabolismo , Ratones , PPAR gamma/agonistas , Pioglitazona/farmacología , Proteinosis Alveolar Pulmonar/metabolismo , Transducción de Señal/efectos de los fármacos
7.
J Immunol ; 178(3): 1645-53, 2007 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-17237414

RESUMEN

CD40-CD154 interaction is pivotal for cell-mediated immunity. There are contradictory reports on whether HIV-1 infection impairs CD154 induction. The interaction between CD40 and CD154 is important not only because it results in activation of APCs but also because it controls CD154 by diminishing expression of this molecule. Compared with healthy controls, CD4(+) T cells from HIV-1(+) patients had impaired induction of CD154 when T cell activation was mediated by CD40(+) APCs. In contrast, T cell activation in the absence of these cells resulted in normal CD154 expression. CD154 induction in HIV-1(+) patients and controls were similar upon blockade of CD40-CD154 binding. Defective regulation of CD154 appeared to occur downstream of the control of mRNA levels because up-regulation of CD154 mRNA was not impaired by HIV-1 infection. This work identifies CD40 as a mediator of impaired CD154 induction in HIV-1 infection and explains why this defect was not detected by studies where T cell activation was triggered independently of CD40(+) APCs. In addition, dysregulation of CD154 in HIV-1 infection likely contributes to immunodeficiency because diminished expression of CD154 induced by CD40 is of functional relevance, resulting in decreased dendritic cell maturation.


Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Antígenos CD40/fisiología , Ligando de CD40/genética , Infecciones por VIH/inmunología , Células Presentadoras de Antígenos/inmunología , Linfocitos T CD4-Positivos/virología , Estudios de Casos y Controles , Células Dendríticas/citología , Regulación hacia Abajo , Regulación de la Expresión Génica , Humanos , Inmunidad Celular , Activación de Linfocitos , ARN Mensajero/análisis
8.
Autophagy ; 3(3): 245-8, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17224624

RESUMEN

A fundamental question in host-pathogen interaction is to determine if the immune system activates fusion with the lysosomes to eradicate pathogens. We recently reported that this task is accomplished by the interaction between CD40 expressed on macrophages and CD154 expressed on activated CD4+ T cells. CD40 stimulation of macrophages induces vacuole-lysosome fusion through autophagy and results in killing of the obligate intracellular pathogen Toxoplasma gondii. This response is independent of IFN-gamma, STAT1 and p47 GTPases. We now report that vacuole-lysosome fusion is dependent on synergy between TRAF6 signaling downstream of CD40 and TNF-alpha. These studies identified a new paradigm by which T cells eradicate an intracellular pathogen within macrophages.


Asunto(s)
Autofagia/inmunología , Antígenos CD40/inmunología , Macrófagos/inmunología , Linfocitos T/inmunología , Factor 6 Asociado a Receptor de TNF/metabolismo , Toxoplasma/inmunología , Secuencia de Aminoácidos , Animales , Antígenos CD40/genética , Ligando de CD40/genética , Ligando de CD40/inmunología , Humanos , Lisosomas/metabolismo , Macrófagos/citología , Macrófagos/parasitología , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismo , Vacuolas/metabolismo
9.
Infect Immun ; 74(3): 1573-9, 2006 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-16495528

RESUMEN

CD40-CD154 interaction is pivotal for resistance against numerous pathogens. However, it is not known if this pathway can also enhance in vivo resistance in gamma interferon (IFN-gamma)-deficient hosts. This is an important question because patients and mice with defects in type 1 cytokine response can control a variety of pathogens. While blockade of endogenous CD154 resulted in a remarkable increase in parasite load in IFN-gamma-/- mice infected with Toxoplasma gondii, in vivo administration of a stimulatory anti-CD40 monoclonal antibody markedly reduced parasite load. This latter effect took place even in T-cell-depleted mice and was accompanied by induction of macrophage toxoplasmacidal activity. CD40 stimulation restricted T. gondii replication independently of STAT1, p47 GTPases, and nitric oxide. In vivo CD40 ligation enhanced tumor necrosis factor alpha (TNF-alpha) production by T. gondii-infected macrophages. In addition, CD40 stimulation required the presence of TNF receptor 2 to reduce parasite load in vivo. These results suggest that CD40-CD154 interaction regulates IFN-gamma-independent mechanisms of host protection through induction of macrophage antimicrobial activity and modulation of TNF-alpha signaling.


Asunto(s)
Antígenos CD40/farmacología , Interferón gamma/metabolismo , Toxoplasma/efectos de los fármacos , Toxoplasmosis Animal/inmunología , Animales , Antígenos CD40/inmunología , Interferón gamma/inmunología , Ratones , Toxoplasma/crecimiento & desarrollo , Toxoplasma/inmunología
10.
Infect Immun ; 73(5): 3115-23, 2005 May.
Artículo en Inglés | MEDLINE | ID: mdl-15845519

RESUMEN

Gamma interferon (IFN-gamma) is the major inducer of classical activation of macrophages. Classically activated mouse macrophages acquire antimicrobial activity that is largely dependent on the production of reactive nitrogen intermediates. However, protection against important intracellular pathogens can take place in the absence of IFN-gamma and nitric oxide synthase 2 (NOS2). Using Toxoplasma gondii as a model, we investigated if CD40 signaling generates mouse macrophages with effector function against an intracellular pathogen despite the absence of priming with IFN-gamma and lack of production of reactive nitrogen intermediates. CD40-stimulated macrophages acquired anti-T. gondii activity that was not inhibited by a neutralizing anti-IFN-gamma monoclonal antibody but was ablated by the neutralization of tumor necrosis factor alpha (TNF-alpha). Moreover, while the induction of anti-T. gondii activity in response to CD40 stimulation was unimpaired in macrophages from IFN-gamma(-/-) mice, macrophages from TNF receptor 1/2(-/-) mice failed to respond to CD40 engagement. In contrast to IFN-gamma-lipopolysaccharide, CD40 stimulation did not induce NOS2 expression and did not trigger production of reactive nitrogen intermediates. Neither N(G)-monomethyl-l-arginine nor diphenyleneiodonium chloride affected the induction of anti-T. gondii activity in response to CD40. Finally, macrophages from NOS2(-/-) mice acquired anti-T. gondii activity in response to CD40 stimulation that was similar to that of macrophages from wild-type mice. These results demonstrate that CD40 induces the antimicrobial activity of macrophages against an intracellular pathogen despite the lack of two central features of classically activated macrophages: priming with IFN-gamma and production of reactive nitrogen intermediates.


Asunto(s)
Antígenos CD40/metabolismo , Interferón gamma/metabolismo , Macrófagos Peritoneales/inmunología , Óxido Nítrico/metabolismo , Transducción de Señal , Toxoplasma/patogenicidad , Animales , Femenino , Activación de Macrófagos , Macrófagos Peritoneales/parasitología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Especies de Nitrógeno Reactivo/metabolismo , Organismos Libres de Patógenos Específicos , Toxoplasma/inmunología , Toxoplasmosis/inmunología , Toxoplasmosis/parasitología , Factor de Necrosis Tumoral alfa/metabolismo
11.
J Immunol ; 175(9): 6014-21, 2005 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-16237096

RESUMEN

IFN-gamma is considered an essential stimulus that allows macrophages to acquire activity against intracellular pathogens in response to a second signal such as TNF-alpha. However, protection against important pathogens can take place in the absence of IFN-gamma through mechanisms that are still dependent on TNF-alpha. Engagement of CD40 modulates antimicrobial activity in macrophages. However, it is not known whether CD40 can replace IFN-gamma as priming signal for induction of this response. We show that CD40 primes mouse macrophages to acquire antimicrobial activity in response to TNF-alpha. The effect of CD40 was not caused by modulation of IL-10 and TGF-beta production or TNFR expression and did not require IFN-alphabeta signaling. Induction of antimicrobial activity required cooperation between TNFR-associated factor 6-dependent CD40 signaling and TNFR2. These results support a paradigm where TNFR-associated factor 6 signaling downstream of CD40 alters the pattern of response of macrophages to TNF-alpha leading to induction of antimicrobial activity.


Asunto(s)
Antígenos CD40/fisiología , Macrófagos/inmunología , Transducción de Señal/fisiología , Factor 6 Asociado a Receptor de TNF/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Animales , Ligando de CD40/farmacología , Femenino , Interferón gamma/farmacología , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Receptores Tipo II del Factor de Necrosis Tumoral/fisiología , Factor de Crecimiento Transformador beta/biosíntesis
12.
J Immunol ; 171(12): 6750-6, 2003 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-14662879

RESUMEN

Protection against certain intracellular pathogens can take place in the absence of IFN-gamma through mechanisms dependent on TNF-alpha. In this regard, patients with partial defect in IFN-gamma receptor 1 are not susceptible to toxoplasmosis. Thus, we used a model of Toxoplasma gondii infection to investigate whether CD154 modulates IFN-gamma-independent mechanisms of host protection. Human monocyte-derived macrophages treated with recombinant CD154 exhibited increased anti-T. gondii activity. The number of tachyzoites per 100 macrophages at 20 h postinfection was lower in CD154-treated macrophages compared with controls. This was accompanied by a decrease in the percentage of infected cells in CD154-treated macrophages at 20 h compared with 1 h postinfection. CD154-bearing cells also induced antimicrobial activity in T. gondii-infected macrophages. CD154 enhanced macrophage anti-T. gondii activity independently of IFN-gamma. TNF-alpha mediated the effects of CD154 on macrophage anti-T. gondii activity. CD154 increased TNF-alpha production by T. gondii-infected macrophages, and neutralization of TNF-alpha inhibited the effect of CD154 on macrophage anti-T. gondii activity. These results demonstrate that CD154 triggers TNF-alpha-dependent antimicrobial activity in macrophages and suggest that CD154 regulates the mechanisms of host protection that take place when IFN-gamma signaling is deficient.


Asunto(s)
Ligando de CD40/fisiología , Interferón gamma/deficiencia , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Macrófagos/parasitología , Toxoplasma/crecimiento & desarrollo , Toxoplasma/inmunología , Factor de Necrosis Tumoral alfa/fisiología , Adyuvantes Inmunológicos/fisiología , Animales , Células Cultivadas , Relación Dosis-Respuesta Inmunológica , Humanos , Interferón gamma/fisiología , Macrófagos/metabolismo , Monocitos/inmunología , Monocitos/parasitología , Proteínas Recombinantes/farmacología , Transducción de Señal/inmunología , Factor de Necrosis Tumoral alfa/metabolismo
13.
J Infect Dis ; 189(1): 61-70, 2004 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-14702154

RESUMEN

The pathogenesis of immunodeficiency associated with human immunodeficiency virus (HIV) infection remains incompletely understood. CD154, a molecule that is expressed primarily on activated CD4(+) T cells, is pivotal for regulation of cell-mediated and humoral immunity and is crucial for control of many opportunistic infections. We investigated whether CD4(+) T cells from HIV-infected patients exhibit defective induction of CD154 in response to opportunistic pathogens. Incubation of purified human CD4(+) T cells with monocytes plus antigenic preparations of either Candida albicans, cytomegalovirus, or Toxoplasma gondii resulted in induction of CD154. Expression of CD154 in response to these pathogens was impaired in CD4(+) T cells from HIV-infected patients. This defect correlated with decreased production of interleukin (IL)-12 and interferon (IFN)-gamma in response to T. gondii. Recombinant CD154 partially restored secretion of IL-12 and IFN-gamma in response to T. gondii in cells from HIV-infected patients. Together, defective induction of CD154 is likely to contribute to impaired cell-mediated immunity against opportunistic pathogens in HIV-infected patients.


Asunto(s)
Infecciones Oportunistas Relacionadas con el SIDA/etiología , Linfocitos T CD4-Positivos/inmunología , Ligando de CD40/biosíntesis , Infecciones por VIH/inmunología , Animales , Antígenos CD/biosíntesis , Antígenos de Diferenciación de Linfocitos T/biosíntesis , Antígenos Fúngicos/farmacología , Antígenos de Protozoos/farmacología , Linfocitos T CD4-Positivos/efectos de los fármacos , Ligando de CD40/farmacología , Candida albicans/inmunología , Candida albicans/patogenicidad , Células Cultivadas , Humanos , Inmunidad Celular , Interferón gamma/biosíntesis , Interleucina-12/biosíntesis , Lectinas Tipo C , Monocitos/efectos de los fármacos , Monocitos/inmunología , Proteínas Recombinantes/farmacología , Toxoplasma/inmunología , Toxoplasma/patogenicidad
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