Cultivation of Cryptosporidium pawum in a non-adherent human monocytic cell line

International audienceCryptosporidium parvum is a coccidian parasite responsible for severe diarrhea in immunocompromised patients. At present, only therapies of limited efficacies are available and despite recent improvements, an easy to perform and reproducible in vitro model is still lacking. The present study shows that the human monocyte cell line THP-1 supports the growth of C. parvum. After inoculation with oocysts, all the asexual and sexual developmental stages were found. Immunofluorescence controls showed that few oocysts remained after infection, disappeared within the first 24 h and that sexual stages reappeared after the second day postinoculation. A continuous asexual life-cycle proceeded throughout the experiments, with at least E-day cultures. Ultrastructural studies evidenced that the parasites were usually localized at the cell surface and also in the cytoplasm, a feature found in vivo with M cells and macrophages. This culture system is easy to initiate and to maintain with adjustable parasitemias and duration which will allow time-dependent drug-testing and also facilitate the study of the biology of this parasite

Use of Percoll for the infection of cells in vitro with Cryptosporidium parvum oocysts

International audienceA method for the infection of non-adherent THP-1 cells and adherent MDBK cells with Cryptosporidium parÕum oocysts using isotonic Percoll solutions was developed. Excystation was maximal after 2 h, but toxicity increased with the oocystrcell ratio and the incubation time. The infection rates did not increase with the oocystrcell ratio and both cell types were equally parasitized.

Effects of purine nucleosides on the in vitro growth of Cryptosporidium parvum

International audienceThe effect of purine nucleosides on the in vitro growth of Cryptosporidium parvum was studied. Culturing the parasite in THP-1 cells for 72 h in growth medium supplemented with adenosine or inosine improved the parasite yields especially in the first 48 h. Similar results were obtained with parasites cultured in Madin^Darby bovine kidney cells and incubated for 24 h with inosine. The addition of inosine to 72-h cultures enhanced the growth of C. parvum in THP-1 cells, especially the trophic stages, whereas the analogue formycin B was toxic to the parasites and induced a marked decrease in the gamont stages. The monitoring of the added purine nucleosides by high performance liquid chromatography showed that at 37 ‡C in the presence of THP-1 cells, a rapid uptake of inosine occurred with hypoxanthine being the main purine present after 2 h in the medium

Use of a non-adherent cell culture system for testing the effect of 2',3'-dideoxyinosine against Cryptosporidium parvum

International audienceThe in vitro cultivation of Cryptosporidium parvum in the non-adherent cell line THP-1 was evaluated for its capability as a useful additional model to investigate the effect of drugs on this parasite. The purine analog antiviral 2',3'-dideoxyinosine (ddI) was evaluated and compared to the reference molecule paromomycin in sequential 24 hour experiments beginning at 24 and 72 hour post-infection. The ability of this technique to evaluate the various parasite stages showed that ddI displayed a dose-dependent efficacy especially on the trophozoite and sexual stages. Paromomycin displayed a lower efficacy than previously reported. Both drugs induced a decrease in the number of multiparasitized cells. These results indicate that the purine salvage pathway should be a key chemotherapeutic target against C. parvum

Neonate Intestinal Immune Response to CpG Oligodeoxynucleotide Stimulation

Background: The development of mucosal vaccines is crucial to efficiently control infectious agents for which mucosae are the primary site of entry. Major drawbacks of these protective strategies are the lack of effective mucosal adjuvant. Synthetic oligodeoxynucleotides that contain several unmethylated cytosine-guanine dinucleotide (CpG-ODN) motifs are now recognized as promising adjuvants displaying mucosal adjuvant activity through direct activation of TLR9-expressing cells. However, little is known about the efficacy of these molecules in stimulating the intestinal immune system in neonates. Methodology/Principal Findings: First, newborn mice received CpG-ODN orally, and the intestinal cytokine and chemokine response was measured. We observed that oral administration of CpG-ODN induces CXC and CC chemokine responses and a cellular infiltration in the intestine of neonates as detected by immunohistochemistry. We next compared the efficiency of the oral route to intraperitoneal administration in stimulating the intestinal immune responses of both adults and neonates. Neonates were more responsive to TLR9-stimulation than adults whatever the CpG-ODN administration route. Their intestinal epithelial cells (IECs) indirectly responded to TLR9 stimulation and contributed to the CXC chemokine response, whereas other TLR9-bearing cells of the lamina-propria produced CC chemokines and Th1-type cytokines. Moreover, we showed that the intestine of adult exhibited a significantly higher level of IL10 at homeostasis than neonates, which might be responsible for the unresponsiveness to TLR9-stimulation, as confirmed by our findings in IL10-deficient mice. Conclusions/Significance: This is the first report that deciphers the role played by CpG-ODN in the intestine of neonates. This work clearly demonstrates that an intraperitoneal administration of CpG-ODN is more efficient in neonates than in adults to stimulate an intestinal chemokine response due to their lower IL-10 intestinal level. In addition we report the efficiency of the oral route at inducing intestinal chemokine responses in neonate that might be taken into consideration for further vaccine development against neonatal diseases

Essai du vaccin Paracox (ND) dans le controle de la coccidiose chez le poulet jaune

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In vitro cultivation of Cryptosporidium parvum in the non-adherent human monocytic THP-1 cell line

International audienceThis study shows that the human monocytic cell line THP-1 supports the growth of C. parvum. Immunofluorescence controls showed that only scarce oocysts remained after infection and disappeared Within the first 24 h of culture. A continuous asexual life cycle proceeded throughout the experiments, with at least 15d cultures. This model provides a useful tool for studies on the biology of C. parvum in cells involved in its transport in immunocompromised hos