scholarly journals RNA-based therapy for Cryptosporidium parvum infection: proof-of-concept studies

2022 ◽  
Author(s):  
Alejandro Castellanos-Gonzalez ◽  
Aygul Sadiqova ◽  
Justine Ortega-Mendez ◽  
A. Clinton White

Cryptosporidium is a leading cause of moderate-to-severe diarrhea in children. Nitazoxanide, the only FDA-approved treatment for cryptosporidiosis, has limited efficacy in those at highest risk for sequelae. RNA-argonaute (Ago) complexes to Cryptosporidium nucleoside diphosphate kinase (cpNDK) decreased the Cryptosporidium parvum mRNA by 95% in infected cells in vitro. Treatment of mice by oral gavage with ssRNA/Ago complexes encapsulated in lipid nanoparticles led to delivery of the complexes into intestinal epithelial cells. Treatment of C. parvum infected mice with ssRNA/Ago complexes targeting cpNDK led to the resolution of oocyst shedding in 4/5 SCID/beige mice. These results confirm the potential use of antisense therapy as an alternative approach to cryptosporidiosis treatment.

2021 ◽  
Vol 15 ◽  
Author(s):  
Pedro Pozo-Jimenez ◽  
Javier Lucas-Romero ◽  
Jose A. Lopez-Garcia

As multielectrode array technology increases in popularity, accessible analytical tools become necessary. Simultaneous recordings from multiple neurons may produce huge amounts of information. Traditional tools based on classical statistics are either insufficient to analyze multiple spike trains or sophisticated and expensive in computing terms. In this communication, we put to the test the idea that AI algorithms may be useful to gather information about the effective connectivity of neurons in local nuclei at a relatively low computing cost. To this end, we decided to explore the capacity of the algorithm C5.0 to retrieve information from a large series of spike trains obtained from a simulated neuronal circuit with a known structure. Combinatory, iterative and recursive processes using C5.0 were built to examine possibilities of increasing the performance of a direct application of the algorithm. Furthermore, we tested the applicability of these processes to a reduced dataset obtained from original biological recordings with unknown connectivity. This was obtained in house from a mouse in vitro preparation of the spinal cord. Results show that this algorithm can retrieve neurons monosynaptically connected to the target in simulated datasets within a single run. Iterative and recursive processes can identify monosynaptic neurons and disynaptic neurons under favorable conditions. Application of these processes to the biological dataset gives clues to identify neurons monosynaptically connected to the target. We conclude that the work presented provides substantial proof of concept for the potential use of AI algorithms to the study of effective connectivity.


2008 ◽  
Vol 77 (2) ◽  
pp. 837-849 ◽  
Author(s):  
Jin Liu ◽  
Mingqi Deng ◽  
Cheryl A. Lancto ◽  
Mitchell S. Abrahamsen ◽  
Mark S. Rutherford ◽  
...  

ABSTRACT The impact of Cryptosporidium parvum infection on host cell gene expression was investigated by microarray analysis with an in vitro model using human ileocecal HCT-8 adenocarcinoma cells. We found changes in 333 (2.6%) transcripts at at least two of the five (6, 12, 24, 48, and 72 h) postinfection time points. Fifty-one of the regulated genes were associated with apoptosis and were grouped into five clusters based on their expression patterns. Early in infection (6 and 12 h), genes with antiapoptotic roles were upregulated and genes with apoptotic roles were downregulated. Later in infection (24, 48, and 72 h), proapoptotic genes were induced and antiapoptotic genes were downregulated, suggesting a biphasic regulation of apoptosis: antiapoptotic state early and moderately proapoptotic state late in infection. This transcriptional profile matched the actual occurrence of apoptosis in the infected cultures. Apoptosis was first detected at 12 h postinfection and increased to a plateau at 24 h, when 20% of infected cells showed nuclear condensation. In contrast, experimental silencing of Bcl-2 induced apoptosis in 50% of infected cells at 12 h postinfection. This resulted in a decrease in the infection rate and a reduction in the accumulation of meront-containing cells. To test the significance of the moderately proapoptotic state late in the infection, we inhibited apoptosis using pancaspase inhibitor Z-VAD-FMK. This treatment also affected the progression of C. parvum infection, as reinfection, normally seen late (24 h to 48 h), did not occur and accumulation of mature meronts was impaired. Control of host apoptosis is complex and crucial to the life of C. parvum. Apoptosis control has at least two components, early inhibition and late moderate promotion. For a successful infection, both aspects appear to be required.


2017 ◽  
Vol 92 (1) ◽  
Author(s):  
Adrish Sen ◽  
Ayushi Sharma ◽  
Harry B. Greenberg

ABSTRACTSTAT1 phosphorylation in response to exogenous interferon (IFN) administration can be inhibited by rotaviral replication bothin vitroandin vivo. In addition many rotavirus strains are resistant to the actions of different IFN types. The regulation by rotaviruses (RVs) of antiviral pathways mediated by multiple IFN types is not well understood. In this study, we find that during infectionin vitroandin vivo, RVs significantly deplete IFN type I, II, and III receptors (IFNRs). Regulation of IFNRs occurred exclusively within RV-infected cells and could be abrogated by inhibiting the lysosomal-endosomal degradation pathway.In vitro, IFNR degradation was conserved across multiple RV strains that differ in their modes of regulating IFN induction. In suckling mice, exogenously administered type I, II, or III IFN induced phosphorylation of STAT1-Y701 within intestinal epithelial cells (IECs) of suckling mice. Murine EW strain RV infection transiently activated intestinal STAT1 at 1 day postinfection (dpi) but not subsequently at 2 to 3 dpi. In response to injection of purified IFN-α/β or -λ, IECs in EW-infected mice exhibited impaired STAT1-Y701 phosphorylation, correlating with depletion of different intestinal IFNRs and impaired IFN-mediated transcription. The ability of EW murine RV to inhibit multiple IFN types led us to test protection of suckling mice from endotoxin-mediated shock, an outcome that is dependent on the host IFN response. Compared to mortality in controls, mice infected with EW murine RV were substantially protected against mortality following parenteral endotoxin administration. These studies identify a novel mechanism of IFN subversion by RV and reveal an unexpected protective effect of RV infection on endotoxin-mediated shock in suckling mice.IMPORTANCEAntiviral functions of types I, II, and III IFNs are mediated by receptor-dependent activation of STAT1. Here, we find that RV degrades the types I, II, and III IFN receptors (IFNRs)in vitro. In a suckling mouse model, RV effectively blocked STAT1 activation and transcription following injection of different purified IFNs. This correlated with significantly decreased protein expression of intestinal types I and II IFNRs. Recent studies demonstrate that in mice lipopolysaccharide (LPS)-induced lethality is prevented by genetic ablation of IFN signaling genes such as IFNAR1 and STAT1. When suckling mice were infected with RV, they were substantially protected from lethal exposure to endotoxin. These findings provide novel insights into the mechanisms underlying rotavirus regulation of different interferons and are likely to stimulate new research into both rotavirus pathogenesis and endotoxemia.


1998 ◽  
Vol 66 (2) ◽  
pp. 603-607 ◽  
Author(s):  
Mary Cosyns ◽  
Svetlana Tsirkin ◽  
Michelle Jones ◽  
Richard Flavell ◽  
Hitoshi Kikutani ◽  
...  

ABSTRACT Mice with disrupted genes for CD40 and CD40 ligand (CD40L) are unable to clear infection with Cryptosporidium parvum and develop cholangitis. Parasites are present in the gut, gall bladder, and biliary tree, and biliary epithelial cells express CD40 on the cell surface. SCID mice infected with C. parvum for >1 month can clear the infection after reconstitution with spleen cells from CD40, but not CD40L, knockout mice. In an in vitro model, C. parvum-infected HepG2 cells were triggered to apoptosis when incubated with a CD40L-CD8 fusion protein. The requirement for CD40-CD40L interactions for immunity to C. parvum indicated by our results may entail the triggering of apoptosis in infected cells, in addition to the known role of CD40L-CD40 interactions in stimulating cytokine production and promoting T-cell responses.


2019 ◽  
Vol 7 (10) ◽  
pp. 452 ◽  
Author(s):  
Xu ◽  
Guo ◽  
Li ◽  
Zhang ◽  
Wu ◽  
...  

Cryptosporidium parvum is a protozoan parasite that can cause moderate-to-severe diarrhea. Insulinase-like proteases (INS) are one of the largest protein families within the small proteome of the pathogen. However, their roles in C. parvum biology remain un-elucidated. In this study, a member of the protein family, INS-15 of C. parvum encoded by cgd3_4260, was cloned, expressed and characterized to understand its function. INS-15 and its domain I were expressed in Escherichia coli and polyclonal antibodies against the domain I and one specific polypeptide were prepared in rabbits. The role of INS-15 protein in the C. parvum invasion was preliminarily studied. Recombinant INS-15 protein and its domain I were successfully expressed in E. coli, together with various degraded products. The cgd3_4260 gene had a peak expression at 2 h of in vitro C. parvum culture, while the INS-15 protein was expressed in the mid-anterior region of sporozoites and the area of merozoites opposite to the nucleus. Anti-INS-15 domain I antibodies reduced the invasion of C. parvum sporozoites by over 40%. The anterior location of INS-15 in invasion stages and partial reduction of in vitro growth indicate that INS-15 plays some roles in the invasion or early development of C. parvum.


2000 ◽  
Vol 68 (3) ◽  
pp. 1710-1713 ◽  
Author(s):  
Declan F. McCole ◽  
Lars Eckmann ◽  
Fabrice Laurent ◽  
Martin F. Kagnoff

ABSTRACT Cryptosporidium parvum induces moderate levels of apoptosis of cultured human intestinal epithelial cells, which are maximal at 24 h after infection. Apoptosis is further increased inC. parvum-infected cells by inhibition of NF-κB. C. parvum infection also attenuates epithelial apoptosis induced by strongly proapoptotic agents. The data suggest C. parvumhas developed strategies to limit apoptosis in order to facilitate its growth and maturation in the early period after epithelial cell infection.


mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
pp. e02582-20
Author(s):  
Kelli L. VanDussen ◽  
Lisa J. Funkhouser-Jones ◽  
Marianna E. Akey ◽  
Deborah A. Schaefer ◽  
Kevin Ackman ◽  
...  

ABSTRACTThe protozoan parasite Cryptosporidium sp. is a leading cause of diarrheal disease in those with compromised or underdeveloped immune systems, particularly infants and toddlers in resource-poor localities. As an enteric pathogen, Cryptosporidium sp. invades the apical surface of intestinal epithelial cells, where it resides in close proximity to metabolites in the intestinal lumen. However, the effect of gut metabolites on susceptibility to Cryptosporidium infection remains largely unstudied. Here, we first identified which gut metabolites are prevalent in neonatal mice when they are most susceptible to Cryptosporidium parvum infection and then tested the isolated effects of these metabolites on C. parvum invasion and growth in intestinal epithelial cells. Our findings demonstrate that medium or long-chain saturated fatty acids inhibit C. parvum growth, perhaps by negatively affecting the streamlined metabolism in C. parvum, which is unable to synthesize fatty acids. Conversely, long-chain unsaturated fatty acids enhanced C. parvum invasion, possibly by modulating membrane fluidity. Hence, gut metabolites, either from diet or produced by the microbiota, influence C. parvum growth in vitro and may also contribute to the early susceptibility to cryptosporidiosis seen in young animals.IMPORTANCECryptosporidium sp. occupies a unique intracellular niche that exposes the parasite to both host cell contents and the intestinal lumen, including metabolites from the diet and produced by the microbiota. Both dietary and microbial products change over the course of early development and could contribute to the changes seen in susceptibility to cryptosporidiosis in humans and mice. Consistent with this model, we show that the immature gut metabolome influenced the growth of Cryptosporidium parvumin vitro. Interestingly, metabolites that significantly altered parasite growth were fatty acids, a class of molecules that Cryptosporidium sp. is unable to synthesize de novo. The enhancing effects of polyunsaturated fatty acids and the inhibitory effects of saturated fatty acids presented in this study may provide a framework for future studies into this enteric parasite’s interactions with exogenous fatty acids during the initial stages of infection.


2021 ◽  
Vol 12 ◽  
Author(s):  
Rui Xu ◽  
Cong Lai ◽  
Fuxian Yang ◽  
Qiang Zhang ◽  
Na Li ◽  
...  

Cryptosporidium parvum is a major cause of moderate-to-severe diarrhea in humans and animals. Its compact genome contains 22 genes encoding divergent insulinase-like proteases (INS), which are poorly characterized. In this study, two small members of this family, INS-21 encoded by cgd7_2080 and INS-23 encoded by cgd5_3400, were cloned, expressed, and characterized to understand their functions. Recombinant INS-21 and INS-23 were expressed in Escherichia coli and polyclonal antibodies against these two proteins were prepared. The cgd7_2080 gene had a high transcription level during 0–2 h of in vitro C. parvum culture, while cgd5_3400 was highly transcribed at 0–6 h. INS-21 was mostly located in the apical region of sporozoites and merozoites whereas INS-23 was found as spots in sporozoites and merozoites. The immunoelectron microscopy confirmed the expression of INS-21 in the apical region of sporozoites while INS-23 appeared to be expressed in the dense granules of sporozoites. The neutralization efficiency was approximately 35%, when the cultures were treated with anti-INS23 antibodies. These results suggest that INS-21 and INS-23 are expressed in different organelles and might have different functions in the development of C. parvum.


Author(s):  
Georgia Wilke ◽  
Yi Wang ◽  
Soumya Ravindran ◽  
Thaddeus Stappenbeck ◽  
William H. Witola ◽  
...  

2004 ◽  
Vol 72 (10) ◽  
pp. 6125-6131 ◽  
Author(s):  
Amna Hashim ◽  
Marguerite Clyne ◽  
Grace Mulcahy ◽  
Donna Akiyoshi ◽  
Rachel Chalmers ◽  
...  

ABSTRACT It has been recognized recently that human cryptosporidiosis is usually caused by Cryptosporidium parvum genotype I (“human” C. parvum), which is not found in animals. Compared to C. parvum genotype II, little is known of the biology of invasion of the human-restricted C. parvum genotype I. The aims of the present study were (i) to explore and compare with genotype II the pathogenesis of C. parvum genotype I infection by using an established in vitro model of infection and (ii) to examine the possibility that host-specific cell tropism determines species restriction among C. parvum genotypes by using a novel ex vivo small intestinal primary cell model of infection. Oocysts of C. parvum genotypes I and II were used to infect HCT-8 cells and primary intestinal epithelial cells in vitro. Primary cells were harvested from human endoscopic small-bowel biopsies and from bovine duodenum postmortem. C. parvum genotype I infected HCT-8 cells with lower efficiency than C. parvum genotype II. Actin colocalization at the host parasite interface and reduction in levels of invasion after treatment with microfilament inhibitors (cytochalasin B and cytochalasin D) were observed for both genotypes. C. parvum genotype II invaded primary intestinal epithelial cells, regardless of the species of origin. In contrast, C. parvum genotype I invaded only human small-bowel cells. The pathogenesis of C. parvum genotype I differs from C. parvum genotype II. C parvum genotype I does not enter primary bovine intestinal cells, suggesting that the species restriction of this genotype is due to host tissue tropism of the infecting isolate.


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