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1.
PLoS One ; 18(11): e0291937, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-38011121

RESUMEN

Single-domain antibodies (sdAbs) derived from Camelidae heavy-chain-only antibodies (also called nanobodies or VHHs) have advantages over conventional antibodies in terms of their small size and stability to pH and temperature extremes, their ability to express well in microbial hosts, and to be functionally multimerized for enhanced properties. For these reasons, VHHs are showing promise as enteric disease therapeutics, yet little is known as to their pharmacokinetics (PK) within the digestive tract. To improve understanding of enteric VHH PK, we investigated the functional and structural stability of monomeric and multimeric camelid VHH-agents following in vitro incubation with intestinal extracts (chyme) from rabbits and pigs or fecal extracts from human sources, and in vivo in rabbits. The results showed that unstructured domains such as epitopic tags and flexible spacers composed of different amino acid sequences were rapidly degraded by enteric proteases while the functional core VHHs were much more stable to these treatments. Individual VHHs were widely variable in their functional stability to GI tract proteases. Some VHH-based agents which neutralize enteric Shiga toxin Stx2 displayed a functional stability to chyme incubations comparable to that of Stx2-neutralizing IgG and IgA mAbs, thus indicating that selected nanobodies can approach the functional stability of conventional immunoglobulins. Enteric PK data obtained from in vitro incubation studies were consistent with similar incubations performed in vivo in rabbit surgical gut loops. These findings have broad implications for enteric use of VHH-based agents, particularly VHH fusion proteins.


Asunto(s)
Camélidos del Nuevo Mundo , Anticuerpos de Dominio Único , Animales , Humanos , Conejos , Porcinos , Cadenas Pesadas de Inmunoglobulina , Anticuerpos Monoclonales , Secuencia de Aminoácidos , Péptido Hidrolasas
2.
Artículo en Inglés | MEDLINE | ID: mdl-30547015

RESUMEN

Within the liver, Plasmodium sporozoites traverse cells searching for a "suitable" hepatocyte, invading these cells through a process that results in the formation of a parasitophorous vacuole (PV), within which the parasite undergoes intracellular replication as a liver stage. It was previously established that two members of the Plasmodium s48/45 protein family, P36 and P52, are essential for productive invasion of host hepatocytes by sporozoites as their simultaneous deletion results in growth-arrested parasites that lack a PV. Recent studies point toward a pathway of entry possibly involving the interaction of P36 with hepatocyte receptors EphA2, CD81, and SR-B1. However, the relationship between P36 and P52 during sporozoite invasion remains unknown. Here we show that parasites with a single P52 or P36 gene deletion each lack a PV after hepatocyte invasion, thereby pheno-copying the lack of a PV observed for the P52/P36 dual gene deletion parasite line. This indicates that both proteins are equally important in the establishment of a PV and act in the same pathway. We created a Plasmodium yoelii P36mCherry tagged parasite line that allowed us to visualize the subcellular localization of P36 and found that it partially co-localizes with P52 in the sporozoite secretory microneme organelles. Furthermore, through co-immunoprecipitation studies in vivo, we determined that P36 and P52 form a protein complex in sporozoites, indicating a concerted function for both proteins within the PV formation pathway. However, upon sporozoite stimulation, only P36 was released as a secreted protein while P52 was not. Our results support a model in which the putatively glycosylphosphatidylinositol (GPI)-anchored P52 may serve as a scaffold to facilitate the interaction of secreted P36 with the host cell during sporozoite invasion of hepatocytes.


Asunto(s)
Hepatocitos/parasitología , Malaria/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Esporozoítos/metabolismo , Animales , Antígenos CD36/metabolismo , Culicidae , Citoplasma/metabolismo , Femenino , Eliminación de Gen , Técnicas de Inactivación de Genes , Glicosilfosfatidilinositoles , Hepatocitos/patología , Ratones , Ratones Endogámicos BALB C , Plasmodium yoelii/metabolismo , Receptor EphA2/metabolismo , Glándulas Salivales/parasitología , Glándulas Salivales/patología
4.
Nat Commun ; 9(1): 1837, 2018 05 09.
Artículo en Inglés | MEDLINE | ID: mdl-29743474

RESUMEN

Malaria liver stages represent an ideal therapeutic target with a bottleneck in parasite load and reduced clinical symptoms; however, current in vitro pre-erythrocytic (PE) models for Plasmodium vivax and P. falciparum lack the efficiency necessary for rapid identification and effective evaluation of new vaccines and drugs, especially targeting late liver-stage development and hypnozoites. Herein we report the development of a 384-well plate culture system using commercially available materials, including cryopreserved primary human hepatocytes. Hepatocyte physiology is maintained for at least 30 days and supports development of P. vivax hypnozoites and complete maturation of P. vivax and P. falciparum schizonts. Our multimodal analysis in antimalarial therapeutic research identifies important PE inhibition mechanisms: immune antibodies against sporozoite surface proteins functionally inhibit liver stage development and ion homeostasis is essential for schizont and hypnozoite viability. This model can be implemented in laboratories in disease-endemic areas to accelerate vaccine and drug discovery research.


Asunto(s)
Antimaláricos/administración & dosificación , Malaria Falciparum/tratamiento farmacológico , Malaria Vivax/tratamiento farmacológico , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium vivax/crecimiento & desarrollo , Animales , Modelos Animales de Enfermedad , Hepatocitos/parasitología , Humanos , Hígado/parasitología , Malaria Falciparum/parasitología , Malaria Vivax/parasitología , Ratones , Plasmodium falciparum/efectos de los fármacos , Plasmodium vivax/efectos de los fármacos , Esquizontes/efectos de los fármacos , Esquizontes/crecimiento & desarrollo , Esporozoítos/efectos de los fármacos , Esporozoítos/crecimiento & desarrollo
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