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1.
Nat Immunol ; 21(7): 790-801, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32424361

RESUMEN

Plasmodium parasite-specific antibodies are critical for protection against malaria, yet the development of long-lived and effective humoral immunity against Plasmodium takes many years and multiple rounds of infection and cure. Here, we report that the rapid development of short-lived plasmablasts during experimental malaria unexpectedly hindered parasite control by impeding germinal center responses. Metabolic hyperactivity of plasmablasts resulted in nutrient deprivation of the germinal center reaction, limiting the generation of memory B cell and long-lived plasma cell responses. Therapeutic administration of a single amino acid to experimentally infected mice was sufficient to overcome the metabolic constraints imposed by plasmablasts and enhanced parasite clearance and the formation of protective humoral immune memory responses. Thus, our studies not only challenge the current model describing the role and function of blood-stage Plasmodium-induced plasmablasts but they also reveal new targets and strategies to improve anti-Plasmodium humoral immunity.


Asunto(s)
Inmunidad Humoral , Malaria/inmunología , Células Plasmáticas/metabolismo , Plasmodium falciparum/inmunología , Adolescente , Adulto , Aminoácidos/administración & dosificación , Aminoácidos/metabolismo , Animales , Anticuerpos Antiprotozoarios/sangre , Anticuerpos Antiprotozoarios/inmunología , Anticuerpos Antiprotozoarios/metabolismo , Antimaláricos/administración & dosificación , ADN Protozoario/aislamiento & purificación , Modelos Animales de Enfermedad , Centro Germinal/citología , Centro Germinal/inmunología , Centro Germinal/metabolismo , Interacciones Huésped-Parásitos/inmunología , Humanos , Malaria/sangre , Malaria/tratamiento farmacológico , Malaria/parasitología , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Nutrientes/metabolismo , Células Plasmáticas/inmunología , Células Plasmáticas/parasitología , Plasmodium falciparum/genética , Plasmodium falciparum/aislamiento & purificación , Prueba de Estudio Conceptual , Adulto Joven
2.
Cell Microbiol ; 23(1): e13270, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-32981231

RESUMEN

The remarkable deformability of red blood cells (RBCs) depends on the viscoelasticity of the plasma membrane and cell contents and the surface area to volume (SA:V) ratio; however, it remains unclear which of these factors is the key determinant for passage through small capillaries. We used a microfluidic device to examine the traversal of normal, stiffened, swollen, parasitised and immature RBCs. We show that dramatic stiffening of RBCs had no measurable effect on their ability to traverse small channels. By contrast, a moderate decrease in the SA:V ratio had a marked effect on the equivalent cylinder diameter that is traversable by RBCs of similar cellular viscoelasticity. We developed a finite element model that provides a coherent rationale for the experimental observations, based on the nonlinear mechanical behaviour of the RBC membrane skeleton. We conclude that the SA:V ratio should be given more prominence in studies of RBC pathologies.


Asunto(s)
Forma de la Célula , Tamaño de la Célula , Deformación Eritrocítica , Eritrocitos/citología , Eritrocitos/fisiología , Capilares/fisiología , Movimiento Celular , Humanos , Dispositivos Laboratorio en un Chip , Modelos Biológicos
3.
BMC Med ; 19(1): 293, 2021 11 22.
Artículo en Inglés | MEDLINE | ID: mdl-34802442

RESUMEN

BACKGROUND: There is a clear need for novel approaches to malaria vaccine development. We aimed to develop a genetically attenuated blood-stage vaccine and test its safety, infectivity, and immunogenicity in healthy volunteers. Our approach was to target the gene encoding the knob-associated histidine-rich protein (KAHRP), which is responsible for the assembly of knob structures at the infected erythrocyte surface. Knobs are required for correct display of the polymorphic adhesion ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1), a key virulence determinant encoded by a repertoire of var genes. METHODS: The gene encoding KAHRP was deleted from P. falciparum 3D7 and a master cell bank was produced in accordance with Good Manufacturing Practice. Eight malaria naïve males were intravenously inoculated (day 0) with 1800 (2 subjects), 1.8 × 105 (2 subjects), or 3 × 106 viable parasites (4 subjects). Parasitemia was measured using qPCR; immunogenicity was determined using standard assays. Parasites were rescued into culture for in vitro analyses (genome sequencing, cytoadhesion assays, scanning electron microscopy, var gene expression). RESULTS: None of the subjects who were administered with 1800 or 1.8 × 105 parasites developed parasitemia; 3/4 subjects administered 3× 106 parasites developed significant parasitemia, first detected on days 13, 18, and 22. One of these three subjects developed symptoms of malaria simultaneously with influenza B (day 17; 14,022 parasites/mL); one subject developed mild symptoms on day 28 (19,956 parasites/mL); and one subject remained asymptomatic up to day 35 (5046 parasites/mL). Parasitemia rapidly cleared with artemether/lumefantrine. Parasitemia induced a parasite-specific antibody and cell-mediated immune response. Parasites cultured ex vivo exhibited genotypic and phenotypic properties similar to inoculated parasites, although the var gene expression profile changed during growth in vivo. CONCLUSIONS: This study represents the first clinical investigation of a genetically attenuated blood-stage human malaria vaccine. A P. falciparum 3D7 kahrp- strain was tested in vivo and found to be immunogenic but can lead to patent parasitemia at high doses. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (number: ACTRN12617000824369 ; date: 06 June 2017).


Asunto(s)
Antimaláricos , Vacunas contra la Malaria , Malaria Falciparum , Malaria , Antimaláricos/uso terapéutico , Arteméter/uso terapéutico , Combinación Arteméter y Lumefantrina/uso terapéutico , Australia , Humanos , Malaria/tratamiento farmacológico , Vacunas contra la Malaria/efectos adversos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/prevención & control , Masculino , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Desarrollo de Vacunas , Vacunas Atenuadas/efectos adversos
4.
Cell Microbiol ; 21(5): e13005, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30634201

RESUMEN

The simian parasite Plasmodium knowlesi causes severe and fatal malaria infections in humans, but the process of host cell remodelling that underpins the pathology of this zoonotic parasite is only poorly understood. We have used serial block-face scanning electron microscopy to explore the topography of P. knowlesi-infected red blood cells (RBCs) at different stages of asexual development. The parasite elaborates large flattened cisternae (Sinton Mulligan's clefts) and tubular vesicles in the host cell cytoplasm, as well as parasitophorous vacuole membrane bulges and blebs, and caveolar structures at the RBC membrane. Large invaginations of host RBC cytoplasm are formed early in development, both from classical cytostomal structures and from larger stabilised pores. Although degradation of haemoglobin is observed in multiple disconnected digestive vacuoles, the persistence of large invaginations during development suggests inefficient consumption of the host cell cytoplasm. The parasite eventually occupies ~40% of the host RBC volume, inducing a 20% increase in volume of the host RBC and an 11% decrease in the surface area to volume ratio, which collectively decreases the ability of the P. knowlesi-infected RBCs to enter small capillaries of a human erythrocyte microchannel analyser. Ektacytometry reveals a markedly decreased deformability, whereas correlative light microscopy/scanning electron microscopy and python-based skeleton analysis (Skan) reveal modifications to the surface of infected RBCs that underpin these physical changes. We show that P. knowlesi-infected RBCs are refractory to treatment with sorbitol lysis but are hypersensitive to hypotonic lysis. The observed physical changes in the host RBCs may underpin the pathology observed in patients infected with P. knowlesi.


Asunto(s)
Membrana Eritrocítica/metabolismo , Eritrocitos/parasitología , Plasmodium knowlesi/ultraestructura , Citoplasma/metabolismo , Citoplasma/ultraestructura , Membrana Eritrocítica/ultraestructura , Eritrocitos/citología , Eritrocitos/ultraestructura , Hemoglobinas/metabolismo , Interacciones Huésped-Parásitos , Humanos , Merozoítos/ultraestructura , Microscopía Electrónica de Rastreo , Presión Osmótica , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/patogenicidad , Plasmodium knowlesi/crecimiento & desarrollo , Plasmodium knowlesi/patogenicidad , Esquizontes/ultraestructura , Trofozoítos/ultraestructura , Vacuolas/metabolismo , Vacuolas/ultraestructura
5.
Chem Rev ; 118(11): 5330-5358, 2018 06 13.
Artículo en Inglés | MEDLINE | ID: mdl-29676564

RESUMEN

New technologies to diagnose malaria at high sensitivity and specificity are urgently needed in the developing world where the disease continues to pose a huge burden on society. Infrared and Raman spectroscopy-based diagnostic methods have a number of advantages compared with other diagnostic tests currently on the market. These include high sensitivity and specificity for detecting low levels of parasitemia along with ease of use and portability. Here, we review the application of vibrational spectroscopic techniques for monitoring and detecting malaria infection. We discuss the role of vibrational (infrared and Raman) spectroscopy in understanding the processes of parasite biology and its application to the study of interactions with antimalarial drugs. The distinct molecular phenotype that characterizes malaria infection and the high sensitivity enabling detection of low parasite densities provides a genuine opportunity for vibrational spectroscopy to become a front-line tool in the elimination of this deadly disease and provide molecular insights into the chemistry of this unique organism.


Asunto(s)
Malaria/diagnóstico , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Espectrometría Raman/métodos , Animales , Eritrocitos/microbiología , Eritrocitos/patología , Hemo/análisis , Hemoproteínas/análisis , Humanos , Plasmodium/crecimiento & desarrollo , Espectroscopía Infrarroja por Transformada de Fourier/instrumentación , Espectrometría Raman/instrumentación , Vibración
6.
PLoS Pathog ; 13(10): e1006659, 2017 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-28985225

RESUMEN

Transmission of malaria parasites relies on the formation of a specialized blood form called the gametocyte. Gametocytes of the human pathogen, Plasmodium falciparum, adopt a crescent shape. Their dramatic morphogenesis is driven by the assembly of a network of microtubules and an underpinning inner membrane complex (IMC). Using super-resolution optical and electron microscopies we define the ultrastructure of the IMC at different stages of gametocyte development. We characterize two new proteins of the gametocyte IMC, called PhIL1 and PIP1. Genetic disruption of PhIL1 or PIP1 ablates elongation and prevents formation of transmission-ready mature gametocytes. The maturation defect is accompanied by failure to form an enveloping IMC and a marked swelling of the digestive vacuole, suggesting PhIL1 and PIP1 are required for correct membrane trafficking. Using immunoprecipitation and mass spectrometry we reveal that PhIL1 interacts with known and new components of the gametocyte IMC.


Asunto(s)
Microtúbulos/metabolismo , Plasmodium falciparum/crecimiento & desarrollo , Desarrollo Sexual/fisiología , Animales , Microscopía Electrónica/métodos , Microtúbulos/ultraestructura , Plasmodium falciparum/ultraestructura , Transporte de Proteínas
7.
Malar J ; 18(1): 312, 2019 Sep 18.
Artículo en Inglés | MEDLINE | ID: mdl-31533836

RESUMEN

BACKGROUND: Anaemia is a major consequence of malaria, caused by the removal of both infected and uninfected red blood cells (RBCs) from the circulation. Complement activation and reduced expression of complement regulatory proteins (CRPs) on RBCs are an important pathogenic mechanism in severe malarial anaemia in both Plasmodium falciparum and Plasmodium vivax infection. However, little is known about loss of CRPs on RBCs during mild malarial anaemia and in low-density infection. METHODS: The expression of CRP CR1, CD55, CD59, and the phagocytic regulator CD47, on uninfected normocytes and reticulocytes were assessed in individuals from two study populations: (1) P. falciparum and P. vivax-infected patients from a low transmission setting in Sabah, Malaysia; and, (2) malaria-naïve volunteers undergoing P. falciparum induced blood-stage malaria (IBSM). For clinical infections, individuals were categorized into anaemia severity categories based on haemoglobin levels. For IBSM, associations between CRPs and haemoglobin level were investigated. RESULTS: CRP expression on RBC was lower in Malaysian individuals with P. falciparum and P. vivax mild malarial anaemia compared to healthy controls. CRP expression was also reduced on RBCs from volunteers during IBSM. Reduction occurred on normocytes and reticulocytes. However, there was no significant association between reduced CRPs and haemoglobin during IBSM. CONCLUSIONS: Removal of CRPs occurs on both RBCs and reticulocytes during Plasmodium infection even in mild malarial anaemia and at low levels of parasitaemia.


Asunto(s)
Anemia/parasitología , Proteínas del Sistema Complemento/genética , Eritrocitos/metabolismo , Malaria Falciparum/complicaciones , Malaria Vivax/complicaciones , Adulto , Proteínas del Sistema Complemento/metabolismo , Eritrocitos/parasitología , Femenino , Humanos , Malaria Falciparum/parasitología , Malaria Vivax/parasitología , Malasia , Masculino , Persona de Mediana Edad , Plasmodium falciparum/fisiología , Plasmodium vivax/fisiología , Adulto Joven
8.
Anal Chem ; 89(10): 5238-5245, 2017 05 16.
Artículo en Inglés | MEDLINE | ID: mdl-28409627

RESUMEN

New diagnostic tools that can detect malaria parasites in conjunction with other diagnostic parameters are urgently required. In this study, Attenuated Total Reflection Fourier transform infrared (ATR-FTIR) spectroscopy in combination with Partial Least Square Discriminant Analysis (PLS-DA) and Partial Least Square Regression (PLS-R) have been applied as a point-of-care test for identifying malaria parasites, blood glucose, and urea levels in whole blood samples from thick blood films on glass slides. The specificity for the PLS-DA was found to be 98% for parasitemia levels >0.5%, but a rather low sensitivity of 70% was achieved because of the small number of negative samples in the model. In PLS-R the Root Mean Square Error of Cross Validation (RMSECV) for parasite concentration (0-5%) was 0.58%. Similarly, for glucose (0-400 mg/dL) and urea (0-250 mg/dL) spiked samples, relative RMSECVs were 16% and 17%, respectively. The method reported here is the first example of multianalyte/disease diagnosis using ATR-FTIR spectroscopy, which in this case, enabled the simultaneous quantification of glucose and urea analytes along with malaria parasitemia quantification using one spectrum obtained from a single drop of blood on a glass microscope slide.


Asunto(s)
Glucosa/química , Malaria/diagnóstico , Plasmodium/citología , Espectroscopía Infrarroja por Transformada de Fourier/métodos , Urea/química , Área Bajo la Curva , Análisis Discriminante , Pruebas con Sangre Seca , Vidrio/química , Humanos , Análisis de los Mínimos Cuadrados , Plasmodium/química , Curva ROC
9.
Analyst ; 142(8): 1192-1199, 2017 Apr 10.
Artículo en Inglés | MEDLINE | ID: mdl-27921101

RESUMEN

Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) has the potential to become a new diagnostic tool for malaria and other diseases. For point-of-care testing, the use of ATR-FTIR in malaria diagnosis enables the analysis of blood in the aqueous state, which represents an enormous advantage by minimising the sample preparation by removing the need for cell fixation. Here we report the quantification of malaria parasitemia in human RBCs in their normal physiological aqueous state. A potential confounding variable for spectroscopic measurements performed on blood are the various anticoagulants that are required to prevent clotting. Accordingly, we tested the effects of 3 common anticoagulants; Sodium Citrate (SC), Potassium Ethylenediaminetetraacetic Acid (EDTA) and lithium heparin on plasma and whole blood in the aqueous and dry phase. Principal Component Analysis (PCA) revealed the model was heavily influenced by the anticoagulants in the case of dry samples, however, in aqueous whole blood samples, the effect was less pronounced as the water in the sample presumably diluted the amount of anticoagulant in contact with the ATR crystal. The possible influence of the anticoagulant effect on the ability to quantify parasitemia levels was tested using Partial Least Squares Regression Analysis (PLS-R). There was no influence of anticoagulants on quantification in the 0-1% range, however attempts to quantify at lower levels (0-0.1%) was best achieved with heparin compared to the other two anticoagulants. The results demonstrate ability to diagnose malaria using ATR-FTIR spectroscopy using wet RBC samples as well as underscoring the desirability to perform wet measurements as these minimise the possible confounding influence of anticoagulants used in blood collection.


Asunto(s)
Anticoagulantes/química , Eritrocitos/parasitología , Malaria/diagnóstico , Parasitemia/diagnóstico , Espectroscopía Infrarroja por Transformada de Fourier , Humanos
10.
Faraday Discuss ; 187: 341-52, 2016 06 23.
Artículo en Inglés | MEDLINE | ID: mdl-27071693

RESUMEN

New highly sensitive tools for malaria diagnostics are urgently needed to enable the detection of infection in asymptomatic carriers and patients with low parasitemia. In pursuit of a highly sensitive diagnostic tool that can identify parasite infections at the single cell level, we have been exploring Fourier transform infrared (FTIR) microscopy using a Focal Plane Array (FPA) imaging detector. Here we report for the first time the application of a new optic configuration developed by Agilent that incorporates 25× condenser and objective Cassegrain optics with a high numerical aperture (NA = 0.81) along with additional high magnification optics within the microscope to provide 0.66 micron pixel resolution (total IR system magnification of 61×) to diagnose malaria parasites at the single cell level on a conventional glass microscope slide. The high quality images clearly resolve the parasite's digestive vacuole demonstrating sub-cellular resolution using this approach. Moreover, we have developed an algorithm that first detects the cells in the infrared image, and secondly extracts the average spectrum. The average spectrum is then run through a model based on Partial Least Squares-Discriminant Analysis (PLS-DA), which diagnoses unequivocally the infected from normal cells. The high quality images, and the fact this measurement can be achieved without a synchrotron source on a conventional glass slide, shows promise as a potential gold standard for malaria detection at the single cell level.


Asunto(s)
Eritrocitos/parasitología , Vidrio/química , Malaria/parasitología , Microscopía/instrumentación , Parásitos/aislamiento & purificación , Análisis de la Célula Individual/métodos , Espectroscopía Infrarroja por Transformada de Fourier/instrumentación , Animales , Humanos , Malaria/diagnóstico , Microscopía/métodos , Parásitos/citología , Plasmodium/citología , Plasmodium/aislamiento & purificación , Análisis de la Célula Individual/instrumentación , Espectroscopía Infrarroja por Transformada de Fourier/métodos
11.
Immunol Cell Biol ; 92(3): 287-97, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24366518

RESUMEN

Most vaccines developed against Chlamydia using animal models provide partial protection against a genital tract infection. However, protection against the oviduct pathology associated with infertility is highly variable and often has no defining immunological correlate. When comparing two adjuvants (CTA1-DD and a combination of Cholera toxin plus CpG-oligodeoxynucleotide-CT/CpG) combined with the chlamydial major outer membrane protein (MOMP) antigen and delivered via the intranasal (IN), sublingual (SL) or transcutaneous (TC) routes, we identified two vaccine groups with contrasting outcomes following infection. SL immunization with MOMP/CTA1-DD induced a 70% reduction in the incidence of oviduct pathology, without significantly altering the course of infection. Conversely, IN immunization with MOMP/CT/CpG prevented an ascending infection, but not the oviduct pathology. This anomaly presented a unique opportunity to study the mechanisms by which vaccines can prevent oviduct pathology, other than by controlling the infection. The IL-17 signaling in the oviducts was found to associate with both the enhancement of immunity to infection and the development of oviduct pathology. This conflicting role of IL-17 may provide some explanation for the discordance in protection between infection and disease and suggests that controlling immunopathology, as opposed to the rapid eradication of the infection, may be essential for an effective human chlamydial vaccine that prevents infertility.


Asunto(s)
Infecciones por Chlamydia/inmunología , Infecciones por Chlamydia/patología , Chlamydia muridarum/inmunología , Inmunidad , Interleucina-17/metabolismo , Transducción de Señal/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Especificidad de Anticuerpos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Separación Celular , Infecciones por Chlamydia/microbiología , Infecciones por Chlamydia/prevención & control , Citocinas/biosíntesis , Femenino , Regulación de la Expresión Génica , Inmunidad/genética , Mediadores de Inflamación/metabolismo , Cinética , Ganglios Linfáticos/patología , Linfocitos/inmunología , Ratones , Infiltración Neutrófila , Oviductos/patología , Bazo/patología , Vacunación , Vagina/inmunología , Vagina/patología
12.
medRxiv ; 2024 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-39108527

RESUMEN

Osteoprotegerin (OPG) is a soluble decoy receptor for receptor activator of NF-ƙB ligand (RANKL) and TNF-related apoptosis-inducing ligand (TRAIL), and is increasingly recognised as a marker of poor prognosis in a number of diseases. Here we demonstrate that in Malaysian adults with falciparum and vivax malaria, OPG is increased, and its ligands TRAIL and RANKL decreased, in proportion to disease severity. In volunteers experimentally infected with P. falciparum and P. vivax, RANKL was suppressed, while TRAIL was unexpectedly increased, suggesting binding of OPG to RANKL prior to TRAIL. We also demonstrate that P. falciparum stimulates B cells to produce OPG in vitro, and that B cell OPG production is increased ex vivo in patients with falciparum, vivax and knowlesi malaria. Our findings provide further evidence of the importance of the OPG/RANKL/TRAIL pathway in pathogenesis of diseases involving systemic inflammation, and may have implications for adjunctive therapies. Further evaluation of the role of B cell production of OPG in host responses to malaria and other inflammatory diseases is warranted.

13.
Commun Biol ; 6(1): 861, 2023 08 18.
Artículo en Inglés | MEDLINE | ID: mdl-37596377

RESUMEN

The malaria parasite uses actin-based mechanisms throughout its lifecycle to control a range of biological processes including intracellular trafficking, gene regulation, parasite motility and invasion. In this work we assign functions to the Plasmodium falciparum formins 1 and 2 (FRM1 and FRM2) proteins in asexual and sexual blood stage development. We show that FRM1 is essential for merozoite invasion and FRM2 is required for efficient cell division. We also observed divergent functions for FRM1 and FRM2 in gametocyte development. Conditional deletion of FRM1 leads to a delay in gametocyte stage progression. We show that FRM2 controls the actin and microtubule cytoskeletons in developing gametocytes, with premature removal of the protein resulting in a loss of transmissible stage V gametocytes. Lastly, we show that targeting formin proteins with the small molecule inhibitor of formin homology domain 2 (SMIFH2) leads to a multistage block in asexual and sexual stage parasite development.


Asunto(s)
Actinas , Plasmodium falciparum , Actinas/genética , Forminas , Plasmodium falciparum/genética , División Celular , Citoesqueleto
14.
Nat Commun ; 14(1): 7387, 2023 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-37968278

RESUMEN

Plasmodium falciparum malaria drives immunoregulatory responses across multiple cell subsets, which protects from immunopathogenesis, but also hampers the development of effective anti-parasitic immunity. Understanding malaria induced tolerogenic responses in specific cell subsets may inform development of strategies to boost protective immunity during drug treatment and vaccination. Here, we analyse the immune landscape with single cell RNA sequencing during P. falciparum malaria. We identify cell type specific responses in sub-clustered major immune cell types. Malaria is associated with an increase in immunosuppressive monocytes, alongside NK and γδ T cells which up-regulate tolerogenic markers. IL-10-producing Tr1 CD4 T cells and IL-10-producing regulatory B cells are also induced. Type I interferon responses are identified across all cell types, suggesting Type I interferon signalling may be linked to induction of immunoregulatory networks during malaria. These findings provide insights into cell-specific and shared immunoregulatory changes during malaria and provide a data resource for further analysis.


Asunto(s)
Interferón Tipo I , Malaria Falciparum , Malaria , Humanos , Interleucina-10/genética , Transcriptoma , Interferón Tipo I/genética , Plasmodium falciparum/genética , Subgrupos de Linfocitos T
15.
J Clin Invest ; 133(19)2023 10 02.
Artículo en Inglés | MEDLINE | ID: mdl-37781920

RESUMEN

The development of highly effective malaria vaccines and improvement of drug-treatment protocols to boost antiparasitic immunity are critical for malaria elimination. However, the rapid establishment of parasite-specific immune regulatory networks following exposure to malaria parasites hampers these efforts. Here, we identified stimulator of interferon genes (STING) as a critical mediator of type I interferon production by CD4+ T cells during blood-stage Plasmodium falciparum infection. The activation of STING in CD4+ T cells by cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) stimulated IFNB gene transcription, which promoted development of IL-10- and IFN-γ-coproducing CD4+ T (type I regulatory [Tr1]) cells. The critical role for type I IFN signaling for Tr1 cell development was confirmed in vivo using a preclinical malaria model. CD4+ T cell sensitivity to STING phosphorylation was increased in healthy volunteers following P. falciparum infection, particularly in Tr1 cells. These findings identified STING expressed by CD4+ T cells as an important mediator of type I IFN production and Tr1 cell development and activation during malaria.


Asunto(s)
Interferón Tipo I , Malaria Falciparum , Linfocitos T Reguladores , Humanos , Linfocitos T CD4-Positivos , Interferón Tipo I/inmunología , Malaria Falciparum/inmunología , Linfocitos T Reguladores/inmunología
16.
Sci Transl Med ; 15(680): eadd9012, 2023 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-36696483

RESUMEN

Natural killer (NK) cells likely play an important role in immunity to malaria, but the effect of repeated malaria on NK cell responses remains unclear. Here, we comprehensively profiled the NK cell response in a cohort of 264 Ugandan children. Repeated malaria exposure was associated with expansion of an atypical, CD56neg population of NK cells that differed transcriptionally, epigenetically, and phenotypically from CD56dim NK cells, including decreased expression of PLZF and the Fc receptor γ-chain, increased histone methylation, and increased protein expression of LAG-3, KIR, and LILRB1. CD56neg NK cells were highly functional and displayed greater antibody-dependent cellular cytotoxicity than CD56dim NK cells. Higher frequencies of CD56neg NK cells were associated with protection against symptomatic malaria and high parasite densities. After marked reductions in malaria transmission, frequencies of these cells rapidly declined, suggesting that continuous exposure to Plasmodium falciparum is required to maintain this modified, adaptive-like NK cell subset.


Asunto(s)
Células Asesinas Naturales , Malaria , Niño , Humanos , Antígeno CD56/metabolismo , Plasmodium falciparum , Receptores Fc
17.
EBioMedicine ; 75: 103784, 2022 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-34968760

RESUMEN

BACKGROUND: Protective malarial antibodies are acquired more rapidly in adults than children, independently of cumulative exposure, however the cellular responses mediating these differences are unknown. CD4 T-follicular helper (Tfh) cells have key roles in inducing antibodies, with Th2-Tfh cell activation associated with antibody development in malaria. Whether Tfh cell activation in malaria is age dependent is unknown and no studies have compared Tfh cell activation in children and adults with malaria. METHODS: We undertook a comprehensive study of Tfh cells, along with B cells and antibody induction in children and adults with malaria. Activation and proliferation of circulating Tfh (cTfh) cell subsets was measured ex vivo and parasite-specific Tfh cell frequencies and functions studied with Activation Induced Marker (AIM) assays and intracellular cytokine staining. FINDINGS: During acute malaria, the magnitude of cTfh cell activation was higher in adults than in children and occurred across all cTfh cell subsets in adults but was restricted only to the Th1-cTfh subset in children. Further, adults had higher levels of parasite-specific cTfh cells, and cTfh cells which produced more Th2-Tfh associated cytokine IL-4. Consistent with a role of higher Tfh cell activation in rapid immune development in adults, adults had higher activation of B cells during infection and higher induction of antibodies 7 and 28 days after malaria compared to children. INTERPRETATION: Our data provide evidence that age impacts Tfh cell activation during malaria, and that these differences may influence antibody induction after treatment. Findings have important implications for vaccine development in children. FUNDING: This word was supported by the National Health and Medical Research Council of Australia, Wellcome Trust, Charles Darwin University Menzies School of Health Research, Channel 7 Children's Research Foundation, and National Health Institute.


Asunto(s)
Malaria Falciparum , Células T Auxiliares Foliculares , Adulto , Australia , Linfocitos B , Niño , Humanos
18.
Nat Commun ; 13(1): 4159, 2022 07 18.
Artículo en Inglés | MEDLINE | ID: mdl-35851033

RESUMEN

T-follicular helper (Tfh) cells are key drivers of antibodies that protect from malaria. However, little is known regarding the host and parasite factors that influence Tfh and functional antibody development. Here, we use samples from a large cross-sectional study of children residing in an area of high malaria transmission in Uganda to characterize Tfh cells and functional antibodies to multiple parasites stages. We identify a dramatic re-distribution of the Tfh cell compartment with age that is independent of malaria exposure, with Th2-Tfh cells predominating in early childhood, while Th1-Tfh cell gradually increase to adult levels over the first decade of life. Functional antibody acquisition is age-dependent and hierarchical acquired based on parasite stage, with merozoite responses followed by sporozoite and gametocyte antibodies. Antibodies are boosted in children with current infection, and are higher in females. The children with the very highest antibody levels have increased Tfh cell activation and proliferation, consistent with a key role of Tfh cells in antibody development. Together, these data reveal a complex relationship between the circulating Tfh compartment, antibody development and protection from malaria.


Asunto(s)
Malaria , Células T Auxiliares Foliculares , Adulto , Anticuerpos Antiprotozoarios , Niño , Preescolar , Estudios Transversales , Femenino , Humanos , Linfocitos T Colaboradores-Inductores , Uganda
19.
Biol Imaging ; 1: e2, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-35036920

RESUMEN

Microscopic examination of blood smears remains the gold standard for laboratory inspection and diagnosis of malaria. Smear inspection is, however, time-consuming and dependent on trained microscopists with results varying in accuracy. We sought to develop an automated image analysis method to improve accuracy and standardization of smear inspection that retains capacity for expert confirmation and image archiving. Here, we present a machine learning method that achieves red blood cell (RBC) detection, differentiation between infected/uninfected cells, and parasite life stage categorization from unprocessed, heterogeneous smear images. Based on a pretrained Faster Region-Based Convolutional Neural Networks (R-CNN) model for RBC detection, our model performs accurately, with an average precision of 0.99 at an intersection-over-union threshold of 0.5. Application of a residual neural network-50 model to infected cells also performs accurately, with an area under the receiver operating characteristic curve of 0.98. Finally, combining our method with a regression model successfully recapitulates intraerythrocytic developmental cycle with accurate lifecycle stage categorization. Combined with a mobile-friendly web-based interface, called PlasmoCount, our method permits rapid navigation through and review of results for quality assurance. By standardizing assessment of Giemsa smears, our method markedly improves inspection reproducibility and presents a realistic route to both routine lab and future field-based automated malaria diagnosis.

20.
mBio ; 11(2)2020 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-32184257

RESUMEN

The malaria parasite Plasmodium falciparum traffics the virulence protein P. falciparum erythrocyte membrane protein 1 (PfEMP1) to the surface of infected red blood cells (RBCs) via membranous organelles, known as the Maurer's clefts. We developed a method for efficient enrichment of Maurer's clefts and profiled the protein composition of this trafficking organelle. We identified 13 previously uncharacterized or poorly characterized Maurer's cleft proteins. We generated transfectants expressing green fluorescent protein (GFP) fusions of 7 proteins and confirmed their Maurer's cleft location. Using co-immunoprecipitation and mass spectrometry, we generated an interaction map of proteins at the Maurer's clefts. We identified two key clusters that may function in the loading and unloading of PfEMP1 into and out of the Maurer's clefts. We focus on a putative PfEMP1 loading complex that includes the protein GEXP07/CX3CL1-binding protein 2 (CBP2). Disruption of GEXP07 causes Maurer's cleft fragmentation, aberrant knobs, ablation of PfEMP1 surface expression, and loss of the PfEMP1-mediated adhesion. ΔGEXP07 parasites have a growth advantage compared to wild-type parasites, and the infected RBCs are more deformable and more osmotically fragile.IMPORTANCE The trafficking of the virulence antigen PfEMP1 and its presentation at the knob structures at the surface of parasite-infected RBCs are central to severe adhesion-related pathologies such as cerebral and placental malaria. This work adds to our understanding of how PfEMP1 is trafficked to the RBC membrane by defining the protein-protein interaction networks that function at the Maurer's clefts controlling PfEMP1 loading and unloading. We characterize a protein needed for virulence protein trafficking and provide new insights into the mechanisms for host cell remodeling, parasite survival within the host, and virulence.


Asunto(s)
Membrana Eritrocítica/metabolismo , Eritrocitos/citología , Interacciones Huésped-Parásitos , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular , Membrana Eritrocítica/parasitología , Eritrocitos/parasitología , Humanos , Proteínas de la Membrana , Plasmodium falciparum/genética , Plasmodium falciparum/patogenicidad , Mapas de Interacción de Proteínas , Transporte de Proteínas , Proteínas Protozoarias/genética
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