Bact-to-Batch: A Microbiota-Based Tool to Determine Optimal Animal Allocation in Experimental Designs.

The basis of any animal experimentation begins with the housing of animals that should take into account the need for splitting animals into similar groups. Even if it is generally recommended to use the minimum number of animals necessary to obtain reliable and statistically significant results (3Rs rule), the allocation of animals is currently mostly based on randomness. Since variability in gut microbiota is an important confounding factor in animal experiments, the main objective of this study was to develop a new approach based on 16S rRNA gene sequencing analysis of the gut microbiota of animals participating in an experiment, in order to correctly assign the animals across batches. For this purpose, a pilot study was performed on 20 mouse faecal samples with the aim of establishing two groups of 10 mice as similar as possible in terms of their faecal microbiota fingerprinting assuming that this approach limits future analytical bias and ensures reproducibility. The suggested approach was challenged with previously published data from a third-party study. This new method allows to embrace the unavoidable microbiota variability between animals in order to limit artefacts and to provide an additional assurance for the reproducibility of animal experiments.

Persistent Cryptosporidium parvum Infection Leads to the Development of the Tumor Microenvironment in an Experimental Mouse Model: Results of a Microarray Approach.

Cryptosporidium spp. are enteric protozoa parasites that infect a variety of vertebrate hosts. These parasites are capable of inducing life-threatening gastrointestinal disease in immunocompromised individuals. With the rising epidemiological evidence of the occurrence of Cryptosporidium infections in humans with digestive cancer, the tumorigenic potential of the parasite has been speculated. In this regard, Cryptosporidium parvum has been reported to induce digestive adenocarcinoma in a rodent model of chronic cryptosporidiosis. However, the processes by which the parasite could induce this carcinogenesis are still unknown. Therefore, the transcriptomes of C. parvum infected ileo-cecal regions of mice developing tumors were analyzed in the current study. For the first time, downregulation of the expression of α-defensin, an anti-microbial target of the parasite in response to C. parvum infection was observed in the transformed tissues. This phenomenon has been speculated to be the result of resistance of C. parvum to the host defense through the upregulated expression of interferon γ-stimulated genes. The inflammatory response generated as result of attenuated expression of anti-microbial peptides highlights the role of immune evasion in the C. parvum-induced tumorigenesis. The study has also succeeded in the characterization of the tumor microenvironment (TME) which is characterized by the presence of cancer associated fibroblasts, myeloid-derived suppressor cells, tumor-associated macrophages and extracellular matrix components. Identification of immune suppressor cells and accumulation of pro-inflammatory mediators speculates that chronic inflammation induced by persistent C. parvum infection assists in development of an immunosuppressive tumor microenvironment.

Genetic basis for virulence differences of various Cryptosporidium parvum carcinogenic isolates.

Cryptosporidium parvum is known to cause life-threatening diarrhea in immunocompromised hosts and was also reported to be capable of inducing digestive adenocarcinoma in a rodent model. Interestingly, three carcinogenic isolates of C. parvum, called DID, TUM1 and CHR, obtained from fecal samples of naturally infected animals or humans, showed higher virulence than the commercially available C. parvum IOWA isolate in our animal model in terms of clinical manifestations, mortality rate and time of onset of neoplastic lesions. In order to discover the potential genetic basis of the differential virulence observed between C. parvum isolates and to contribute to the understanding of Cryptosporidium virulence, entire genomes of the isolates DID, TUM1 and CHR were sequenced then compared to the C. parvum IOWA reference genome. 125 common SNVs corresponding to 90 CDSs were found in the C. parvum genome that could explain this differential virulence. In particular variants in several membrane and secreted proteins were identified. Besides the genes already known to be involved in parasite virulence, this study identified potential new virulence factors whose functional characterization can be achieved through CRISPR/Cas9 technology applied to this parasite.

2,6-Diaminopurine as a highly potent corrector of UGA nonsense mutations.

Nonsense mutations cause about 10% of genetic disease cases, and no treatments are available. Nonsense mutations can be corrected by molecules with nonsense mutation readthrough activity. An extract of the mushroom Lepista inversa has recently shown high-efficiency correction of UGA and UAA nonsense mutations. One active constituent of this extract is 2,6-diaminopurine (DAP). In Calu-6 cancer cells, in which TP53 gene has a UGA nonsense mutation, DAP treatment increases p53 level. It also decreases the growth of tumors arising from Calu-6 cells injected into immunodeficient nude mice. DAP acts by interfering with the activity of a tRNA-specific 2'-O-methyltransferase (FTSJ1) responsible for cytosine 34 modification in tRNATrp. Low-toxicity and high-efficiency UGA nonsense mutation correction make DAP a good candidate for the development of treatments for genetic diseases caused by nonsense mutations.

An Interphase Microfluidic Culture System for the Study of Ex Vivo Intestinal Tissue.

Ex vivo explant culture models offer unique properties to study complex mechanisms underlying tissue growth, renewal, and disease. A major weakness is the short viability depending on the biopsy origin and preparation protocol. We describe an interphase microfluidic culture system to cultivate full thickness murine colon explants which keeps morphological structures of the tissue up to 192 h. The system was composed of a central well on top of a porous membrane supported by a microchannel structure. The microfluidic perfusion allowed bathing the serosal side while preventing immersion of the villi. After eight days, up to 33% of the samples displayed no histological abnormalities. Numerical simulation of the transport of oxygen and glucose provided technical solutions to improve the functionality of the microdevice.

Three-dimensional (3D) culture of adult murine colon as an in vitro model of cryptosporidiosis: Proof of concept.

Cryptosporidium parvum is a major cause of diarrheal illness and was recently potentially associated with digestive carcinogenesis. Despite its impact on human health, Cryptosporidium pathogenesis remains poorly known, mainly due to the lack of a long-term culture method for this parasite. Thus, the aim of the present study was to develop a three-dimensional (3D) culture model from adult murine colon allowing biological investigations of the host-parasite interactions in an in vivo-like environment and, in particular, the development of parasite-induced neoplasia. Colonic explants were cultured and preserved ex vivo for 35 days and co-culturing was performed with C. parvum. Strikingly, the resulting system allowed the reproduction of neoplastic lesions in vitro at 27 days post-infection (PI), providing new evidence of the role of the parasite in the induction of carcinogenesis. This promising model could facilitate the study of host-pathogen interactions and the investigation of the process involved in Cryptosporidium-induced cell transformation.

Cryptosporidium parvum-induced ileo-caecal adenocarcinoma and Wnt signaling in a mouse model.

Cryptosporidium species are apicomplexan protozoans that are found worldwide. These parasites constitute a large risk to human and animal health. They cause self-limited diarrhea in immunocompetent hosts and a life-threatening disease in immunocompromised hosts. Interestingly, Cryptosporidium parvum has been related to digestive carcinogenesis in humans. Consistent with a potential tumorigenic role of this parasite, in an original reproducible animal model of chronic cryptosporidiosis based on dexamethasone-treated or untreated adult SCID mice, we formerly reported that C. parvum (strains of animal and human origin) is able to induce digestive adenocarcinoma even in infections induced with very low inoculum. The aim of this study was to further characterize this animal model and to explore metabolic pathways potentially involved in the development of C. parvum-induced ileo-caecal oncogenesis. We searched for alterations in genes or proteins commonly involved in cell cycle, differentiation or cell migration, such as ß-catenin, Apc, E-cadherin, Kras and p53. After infection of animals with C. parvum we demonstrated immunohistochemical abnormal localization of Wnt signaling pathway components and p53. Mutations in the selected loci of studied genes were not found after high-throughput sequencing. Furthermore, alterations in the ultrastructure of adherens junctions of the ileo-caecal neoplastic epithelia of C. parvum-infected mice were recorded using transmission electron microscopy. In conclusion, we found for the first time that the Wnt signaling pathway, and particularly the cytoskeleton network, seems to be pivotal for the development of the C. parvum-induced neoplastic process and cell migration of transformed cells. Furthermore, this model is a valuable tool in understanding the host-pathogen interactions associated with the intricate infection process of this parasite, which is able to modulate host cytoskeleton activities and several host-cell biological processes and remains a significant cause of infection worldwide.

Cryptosporidium parvum infection in SCID mice infected with only one oocyst: qPCR assessment of parasite replication in tissues and development of digestive cancer.

Dexamethasone (Dex) treated Severe Combined Immunodeficiency (SCID) mice were previously described as developing digestive adenocarcinoma after massive infection with Cryptosporidium parvum as soon as 45 days post-infection (P.I.). We aimed to determine the minimum number of oocysts capable of inducing infection and thereby gastrointestinal tumors in this model. Mice were challenged with calibrated oocyst suspensions containing intended doses of: 1, 10, 100 or 10(5) oocysts of C. parvum Iowa strain. All administered doses were infective for animals but increasing the oocyst challenge lead to an increase in mice infectivity (P = 0.01). Oocyst shedding was detected at 7 days P.I. after inoculation with more than 10 oocysts, and after 15 days in mice challenged with one oocyst. In groups challenged with lower inocula, parasite growth phase was significantly higher (P = 0.005) compared to mice inoculated with higher doses. After 45 days P.I. all groups of mice had a mean of oocyst shedding superior to 10,000 oocyst/g of feces. The most impressive observation of this study was the demonstration that C. parvum-induced digestive adenocarcinoma could be caused by infection with low doses of Cryptosporidium, even with only one oocyst: in mice inoculated with low doses, neoplastic lesions were detected as early as 45 days P.I. both in the stomach and ileo-caecal region, and these lesions could evolve in an invasive adenocarcinoma. These findings show a great amplification effect of parasites in mouse tissues after challenge with low doses as confirmed by quantitative PCR. The ability of C. parvum to infect mice with one oocyst and to develop digestive adenocarcinoma suggests that other mammalian species including humans could be also susceptible to this process, especially when they are severely immunocompromised.

Fulminant cryptosporidiosis after near-drowning: a human Cryptosporidium parvum strain implicated in invasive gastrointestinal adenocarcinoma and cholangiocarcinoma in an experimental model.

In the present work, we report the characterization of a Cryptosporidium parvum strain isolated from a patient who nearly drowned in the Deule River (Lille, France) after being discharged from the hospital where he had undergone allogeneic stem cell transplantation. After being rescued and readmitted to the hospital, he developed fulminant cryptosporidiosis. The strain isolated from the patient's stools was identified as C. parvum II2A15G2R1 (subtype linked to zoonotic exposure) and inoculated into SCID mice. In this host, this virulent C. parvum isolate induced not only severe infection but also invasive gastrointestinal and biliary adenocarcinoma. The observation of adenocarcinomas that progressed through all layers of the digestive tract to the subserosa and spread via blood vessels confirmed the invasive nature of the neoplastic process. These results indicate for the first time that a human-derived C. parvum isolate is able to induce digestive cancer. This study is of special interest considering the exposure of a large number of humans and animals to this waterborne protozoan, which is highly tumorigenic when inoculated in a rodent model.

Fulminant cryptosporidiosis associated with digestive adenocarcinoma in SCID mice infected with Cryptosporidium parvum TUM1 strain.

We recently demonstrated that Cryptosporidium parvum IOWA strain induces in situ ileo-caecal adenocarcinoma in an animal model. Herein, the ability of another C. parvum strain to induce digestive neoplasia in dexamethasone-treated SCID mice was explored. SCID mice infected with C. parvum TUM1 strain developed a fulminant cryptosporidiosis associated with intramucosal adenocarcinoma, which is considered an early histological sign of invasive cancer. Both evidence of a role of C. parvum in adenocarcinoma induction and the extended prevalence of cryptosporidiosis worldwide, suggest that the risk of C. parvum-induced gastro-intestinal cancer in humans should be assessed.

Development of Cryptosporidium parvum-induced gastrointestinal neoplasia in severe combined immunodeficiency (SCID) mice: severity of lesions is correlated with infection intensity.

We reported previously that Cryptosporidium parvum was able to induce intestinal tumors in severe combined immunodeficiency (SCID) mice treated with corticoids. To further characterize this Cryptosporidium-induced cell transformation, SCID mice treated with dexamethasone were challenged with C. parvum oocysts, and euthanatized sequentially after infection for histologic examination. Ki-67 was used as a marker of cellular proliferation. Our previous results were confirmed, and it was also found that mice receiving higher inocula (10(6)-10(7)) experienced more severe neoplastic development. Additionally, neoplastic changes were observed not only in the caecum but also in the stomach and duodenum of some animals. Interestingly, SCID mice (6/6) inoculated with 10(5)-10(7) oocysts showed high grade intraepithelial neoplasia or adenomas with high grade dysplasia in the caecum after Day 46 post-infection (PI). Immunohistochemistry for Ki-67 staining indicated the neoplastic process associated to cryptosporidiosis, and evidenced the first immunohistochemical alterations at early stages of the process, even at 3 weeks PI.

Cryptosporidium parvum, a potential cause of colic adenocarcinoma.

BACKGROUND: Cryptosporidiosis represents a major public health problem. This infection has been reported worldwide as a frequent cause of diarrhoea. Particularly, it remains a clinically significant opportunistic infection among immunocompromised patients, causing potentially life-threatening diarrhoea in HIV-infected persons. However, the understanding about different aspects of this infection such as invasion, transmission and pathogenesis is problematic. Additionally, it has been difficult to find suitable animal models for propagation of this parasite. Efforts are needed to develop reproducible animal models allowing both the routine passage of different species and approaching unclear aspects of Cryptosporidium infection, especially in the pathophysiology field. RESULTS: We developed a model using adult severe combined immunodeficiency (SCID) mice inoculated with Cryptosporidium parvum or Cryptosporidium muris while treated or not with Dexamethasone (Dex) in order to investigate divergences in prepatent period, oocyst shedding or clinical and histopathological manifestations. C. muris-infected mice showed high levels of oocysts excretion, whatever the chemical immunosuppression status. Pre-patent periods were 11 days and 9.7 days in average in Dex treated and untreated mice, respectively. Parasite infection was restricted to the stomach, and had a clear preferential colonization for fundic area in both groups. Among C. parvum-infected mice, Dex-treated SCID mice became chronic shedders with a prepatent period of 6.2 days in average. C. parvum-inoculated mice treated with Dex developed glandular cystic polyps with areas of intraepithelial neoplasia, and also with the presence of intramucosal adenocarcinoma. CONCLUSION: For the first time C. parvum is associated with the formation of polyps and adenocarcinoma lesions in the gut of Dex-treated SCID mice. Additionally, we have developed a model to compare chronic muris and parvum cryptosporidiosis using SCID mice treated with corticoids. This reproducible model has facilitated the evaluation of clinical signs, oocyst shedding, location of the infection, pathogenicity, and histopathological changes in the gastrointestinal tract, indicating divergent effects of Dex according to Cryptosporidium species causing infection.