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Oleylphosphocholine (OlPC) arrests Cryptosporidium parvum growth in vitro and prevents lethal infection in interferon gamma receptor knock-out mice.

Sonzogni-Desautels K, Renteria AE, Camargo FV, Di Lenardo TZ, Mikhail A, Arrowood MJ, Fortin A, Ndao M - Front Microbiol (2015)

Bottom Line: Treatment with OlPC, at 40 mg/kg/day resulted in 100% survival, complete clearance of parasite in stools and a 99.9% parasite burden reduction in the intestines at Day 30.Doses of 30 and 20 mg/kg/day also demonstrated an increased survival rate and a dose-dependent parasite burden reduction.Together, our results strongly support that OlPC represents a potential candidate for the treatment of C. parvum infections in immunocompromised patients.

View Article: PubMed Central - PubMed

Affiliation: National Reference Centre for Parasitology, Research Institute of the McGill University Health Centre, Montreal QC, Canada ; Institute of Parasitology, Macdonald Campus, McGill University, Montreal QC, Canada.

ABSTRACT
Cryptosporidium parvum is a species of protozoa that causes cryptosporidiosis, an intestinal disease affecting many mammals including humans. Typically, in healthy individuals, cryptosporidiosis is a self-limiting disease. However, C. parvum can cause a severe and persistent infection that can be life-threatening for immunocompromised individuals, such as AIDS patients. As there are no available treatments for these patients that can cure the disease, there is an urgent need to identify treatment options. We tested the anti-parasitic activity of the alkylphosphocholine oleylphosphocholine (OlPC), an analog of miltefosine, against C. parvum in in vitro and in vivo studies. In vitro experiments using C. parvum infected human ileocecal adenocarcinoma cells (HCT-8 cells) showed that OlPC has an EC50 of 18.84 nM. Moreover, no cell toxicity has been seen at concentrations ≤50 μM. C57BL/6 interferon gamma receptor knock-out mice, were infected by gavage with 4000 C. parvum oocysts on Day 0. Oral treatments, with OlPC, miltefosine, paromomycin or PBS, began on Day 3 post-infection for 10 days. Treatment with OlPC, at 40 mg/kg/day resulted in 100% survival, complete clearance of parasite in stools and a 99.9% parasite burden reduction in the intestines at Day 30. Doses of 30 and 20 mg/kg/day also demonstrated an increased survival rate and a dose-dependent parasite burden reduction. Mice treated with 10 mg/kg/day of miltefosine resulted in 50% survival at Day 30. In contrast, control mice, treated with PBS or 100 mg/kg/day of paromomycin, died or had to be euthanized between Days 6 and 13 due to severe illness. Results of parasite burden were obtained by qPCR and cross-validated by both flow cytometry of stool oocysts and histological sections of the ileum. Together, our results strongly support that OlPC represents a potential candidate for the treatment of C. parvum infections in immunocompromised patients.

No MeSH data available.


Related in: MedlinePlus

Day 10 histological sections of the ileum of OlPC-treated C57BL/6 IFNγR-KO mice infected with C. parvum. Representative transverse sections of the ileum stained with H&E showing disease progression. (A) Ileum of mice treated with 20 mg/kg/day of OlPC shows significant epithelial damage and abundance of oocysts. (B) Ileum of mice treated with 30 mg/kg/day of OlPC. (C) Ileum of mice treated with 40 mg/kg/day of OlPC. Sections from (B,C) reveal mild gut epithelial damage and rare presence of oocysts. Each group is represented at 100 and 400x magnifications. Red arrows indicate C. parvum oocysts.
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Figure 6: Day 10 histological sections of the ileum of OlPC-treated C57BL/6 IFNγR-KO mice infected with C. parvum. Representative transverse sections of the ileum stained with H&E showing disease progression. (A) Ileum of mice treated with 20 mg/kg/day of OlPC shows significant epithelial damage and abundance of oocysts. (B) Ileum of mice treated with 30 mg/kg/day of OlPC. (C) Ileum of mice treated with 40 mg/kg/day of OlPC. Sections from (B,C) reveal mild gut epithelial damage and rare presence of oocysts. Each group is represented at 100 and 400x magnifications. Red arrows indicate C. parvum oocysts.

Mentions: To visually validate the presence of C. parvum life cycle stages in the intestines of infected mice, the ileum was collected from all mice at time of death. Sections from the ileum were prepared and stained (H&E) for histopathology purposes. Ileum sections from mice treated with PBS and 100 mg/kg/day of paromomycin at Day 10 revealed an overwhelming presence of C. parvum life cycle stages and severe damage to the epithelium of the intestine (Figures 5A,B). Damage to intestinal mucosa includes blunting of the villi, acute inflammation and formation of fibrin (fibrinous hemorrhagic enteritis) resulting from local hemorrhage and clotting (Figures 5A,B). The ileum of mice treated with 10 mg/kg/day of miltefosine was as severely damaged and infected as the other controls (Figure 5C). At Day 10, C. parvum oocysts were also abundantly present in mice treated with 20 mg/kg/day of OlPC and intestinal damage was also present but to a lesser extent than in the infected controls (Figure 6A). At the dose of 30 mg/kg/day of OlPC, mild intestinal damage and inflammation were noticeable and C. parvum oocysts were scarcely visible (Figure 6B). Finally, at 40 mg/kg/day, mice displayed practically no signs of intestinal damage or inflammation and a rare presence of C. parvum oocysts at Day 10 (Figure 6C). Slides from the ileum of non-infected controls were also prepared to provide a negative reference for comparison (data not shown).


Oleylphosphocholine (OlPC) arrests Cryptosporidium parvum growth in vitro and prevents lethal infection in interferon gamma receptor knock-out mice.

Sonzogni-Desautels K, Renteria AE, Camargo FV, Di Lenardo TZ, Mikhail A, Arrowood MJ, Fortin A, Ndao M - Front Microbiol (2015)

Day 10 histological sections of the ileum of OlPC-treated C57BL/6 IFNγR-KO mice infected with C. parvum. Representative transverse sections of the ileum stained with H&E showing disease progression. (A) Ileum of mice treated with 20 mg/kg/day of OlPC shows significant epithelial damage and abundance of oocysts. (B) Ileum of mice treated with 30 mg/kg/day of OlPC. (C) Ileum of mice treated with 40 mg/kg/day of OlPC. Sections from (B,C) reveal mild gut epithelial damage and rare presence of oocysts. Each group is represented at 100 and 400x magnifications. Red arrows indicate C. parvum oocysts.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4585137&req=5

Figure 6: Day 10 histological sections of the ileum of OlPC-treated C57BL/6 IFNγR-KO mice infected with C. parvum. Representative transverse sections of the ileum stained with H&E showing disease progression. (A) Ileum of mice treated with 20 mg/kg/day of OlPC shows significant epithelial damage and abundance of oocysts. (B) Ileum of mice treated with 30 mg/kg/day of OlPC. (C) Ileum of mice treated with 40 mg/kg/day of OlPC. Sections from (B,C) reveal mild gut epithelial damage and rare presence of oocysts. Each group is represented at 100 and 400x magnifications. Red arrows indicate C. parvum oocysts.
Mentions: To visually validate the presence of C. parvum life cycle stages in the intestines of infected mice, the ileum was collected from all mice at time of death. Sections from the ileum were prepared and stained (H&E) for histopathology purposes. Ileum sections from mice treated with PBS and 100 mg/kg/day of paromomycin at Day 10 revealed an overwhelming presence of C. parvum life cycle stages and severe damage to the epithelium of the intestine (Figures 5A,B). Damage to intestinal mucosa includes blunting of the villi, acute inflammation and formation of fibrin (fibrinous hemorrhagic enteritis) resulting from local hemorrhage and clotting (Figures 5A,B). The ileum of mice treated with 10 mg/kg/day of miltefosine was as severely damaged and infected as the other controls (Figure 5C). At Day 10, C. parvum oocysts were also abundantly present in mice treated with 20 mg/kg/day of OlPC and intestinal damage was also present but to a lesser extent than in the infected controls (Figure 6A). At the dose of 30 mg/kg/day of OlPC, mild intestinal damage and inflammation were noticeable and C. parvum oocysts were scarcely visible (Figure 6B). Finally, at 40 mg/kg/day, mice displayed practically no signs of intestinal damage or inflammation and a rare presence of C. parvum oocysts at Day 10 (Figure 6C). Slides from the ileum of non-infected controls were also prepared to provide a negative reference for comparison (data not shown).

Bottom Line: Treatment with OlPC, at 40 mg/kg/day resulted in 100% survival, complete clearance of parasite in stools and a 99.9% parasite burden reduction in the intestines at Day 30.Doses of 30 and 20 mg/kg/day also demonstrated an increased survival rate and a dose-dependent parasite burden reduction.Together, our results strongly support that OlPC represents a potential candidate for the treatment of C. parvum infections in immunocompromised patients.

View Article: PubMed Central - PubMed

Affiliation: National Reference Centre for Parasitology, Research Institute of the McGill University Health Centre, Montreal QC, Canada ; Institute of Parasitology, Macdonald Campus, McGill University, Montreal QC, Canada.

ABSTRACT
Cryptosporidium parvum is a species of protozoa that causes cryptosporidiosis, an intestinal disease affecting many mammals including humans. Typically, in healthy individuals, cryptosporidiosis is a self-limiting disease. However, C. parvum can cause a severe and persistent infection that can be life-threatening for immunocompromised individuals, such as AIDS patients. As there are no available treatments for these patients that can cure the disease, there is an urgent need to identify treatment options. We tested the anti-parasitic activity of the alkylphosphocholine oleylphosphocholine (OlPC), an analog of miltefosine, against C. parvum in in vitro and in vivo studies. In vitro experiments using C. parvum infected human ileocecal adenocarcinoma cells (HCT-8 cells) showed that OlPC has an EC50 of 18.84 nM. Moreover, no cell toxicity has been seen at concentrations ≤50 μM. C57BL/6 interferon gamma receptor knock-out mice, were infected by gavage with 4000 C. parvum oocysts on Day 0. Oral treatments, with OlPC, miltefosine, paromomycin or PBS, began on Day 3 post-infection for 10 days. Treatment with OlPC, at 40 mg/kg/day resulted in 100% survival, complete clearance of parasite in stools and a 99.9% parasite burden reduction in the intestines at Day 30. Doses of 30 and 20 mg/kg/day also demonstrated an increased survival rate and a dose-dependent parasite burden reduction. Mice treated with 10 mg/kg/day of miltefosine resulted in 50% survival at Day 30. In contrast, control mice, treated with PBS or 100 mg/kg/day of paromomycin, died or had to be euthanized between Days 6 and 13 due to severe illness. Results of parasite burden were obtained by qPCR and cross-validated by both flow cytometry of stool oocysts and histological sections of the ileum. Together, our results strongly support that OlPC represents a potential candidate for the treatment of C. parvum infections in immunocompromised patients.

No MeSH data available.


Related in: MedlinePlus