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Try out PMC Labs and tell us what you think. Learn More. Cryptosporidiosis is a leading cause of diarrheal disease and an important contributor to global morbidity and mortality. Although the brunt of disease burden is felt by children in developing countries, Cryptosporidium is a ubiquitous intestinal parasite with frequent outbreaks around the world.

There are no consistently effective treatments for cryptosporidiosis and the research to drive new developments has stagnated, largely due to a lack of efficient in vivo and in vitro models. Fortunately, these research barriers have started to fall. In this review, we highlight two recent advances aiding this process: A tractable mouse model for Cryptosporidium infection and stem cell-based in vitro culture systems that mimic the complexity of the host intestine.

These models are paving the way for researchers to investigate Cryptosporidium infection and host immunity down to a molecular level.

We believe that wise investments made to adopt and develop these new models will reap benefits not only for the Cryptosporidium community but also for the intestinal immunology field at large. Cryptosporidium is a member of the apicomplexan phylum of parasites and a distant relative of Plasmodium , the causative agent of malaria.

Unlike Plasmodium , Cryptosporidium is monoxenous—completing its entire life cycle within a single host—and infection is specifically localized to the gastrointestinal tract. The infectious form of the parasite, known as the oocyst, is resistant to a variety of standard disinfectants, including chlorination.

Each Cryptosporidium oocyst harbors four sporozoites, which are released when oocysts are ingested by a host and invade the apical side of polarized intestinal epithelial cells. Parasitophorous vacuoles can be readily seen lining the intestine of an infected host Figure 1A. Cryptosporidium then undergo asexual and sexual cycles of replication, leading to the production of new oocysts that are shed by the infected host Figure 1B. Cryptosporidium parasites.

A Parasitophorous vacuoles line the wall of the small intestine in an infected mouse. Scanning electron microscopy with parasites in yellow, parasitophorous vacuoles in orange, and host intestinal villi in purple image courtesy of Prof. David Ferguson, Oxford University, copyright retained.

B Life cycle of the Cryptosporidium parasite. Each oocyst contains four sporozoites that can infect the epithelium lining the host intestinal tract. Sporozoites mature to trophozoites and then meronts. Meronts release merozoites which go on to reinvade nearby epithelial cells. The switch from asexual to sexual replication during the Cryptosporidium life cycle is poorly understood and remains an area of great interest. During sexual replication, male and female gametes are produced, and fertilization leads to the formation of new infectious oocysts.

Human cryptosporidiosis is mainly caused by two species: the anthroponotic Cryptosporidium hominis and the zoonotic Cryptosporidium parvum. The pathology of a Cryptosporidium infection manifests as diarrhoeal disease, particularly in children in low-income countries during the first 2 years of life Kotloff et al. While severe cases can be fatal, the majority of Cryptosporidium infections resolve within a few weeks.

Because of this self-limiting nature, it is tempting to focus solely on acute symptoms and short-term consequences. However, infection in young children is closely associated with bouts of prolonged malnutrition that lead to important long-term consequences, such as impaired growth and cognitive development Korpe et al.

A recent re-evaluation of the Cryptosporidium disease burden estimates an annual global loss of nearly 13 million disability adjusted life years; a figure largely driven by malnutrition associated morbidity Khalil et al.

Despite this substantial disease burden, there are no consistently effective treatments for cryptosporidiosis. Nitazoxanide, the only FDA-approved drug to treat infection, has limited efficacy in the immunocompetent and performs on par with placebo in immunocompromised or malnourished patients Amadi et al. Development of new therapeutics has been severely hindered by a lack of effective in vitro and in vivo models for Cryptosporidium infection. However, recent advances in these fields are very promising, with novel platforms for Cryptosporidium research that allow for infection and immunity to be studied in more physiologically-relevant settings.

The genus Cryptosporidium infects a broad range of vertebrate hosts from humans to fish. Despite this wide range of hosts, animal models that recapitulate human infection are historically lacking. Calves, gnotobiotic piglets, and immunocompromised mice have all been used to model infection with varying degrees of success. These in vivo models are still the gold standard of Cryptosporidium infection studies, since they provide the unique dynamic environment of the host intestine, its complex tissue architecture, diversity of cell types, and host immune responses Figure 2A.

However, due to the inability to adequately further manipulate the genetics of these host organisms, and the high costs associated with their upkeep, researchers have been unable to explore mechanisms of the immune response to Cryptosporidium. In an effort to overcome these restrictions, Cryptosporidium tyzzeri , a species of the parasite that naturally infects mice, has recently been developed as a genetically tractable model of human infection Sateriale et al. New models of Cryptosporidium infection.

The use of colours is meant to demonstrate the complexity and diversity of cell types that are present in these models, with the natural mouse model including the host immune response in red. Despite efforts to rescue the original nomenclature, the taxonomic name Cryptosporidium parvum currently refers to the parasite species that is a natural infection of cows, and Cryptosporidium tyzzeri refers to the natural mouse pathogen that Tyzzer likely discovered Ren et al.

Now recognized as a ubiquitous infection of the common house mouse, Mus musculus , the C. Most importantly, C. Because there are no effective drug treatments, resolution of infection is closely tied to the recovery of normal CD4 T-cell counts Wang et al. In the murine model, mice lacking T-cells suffer from protracted and severe infections with C. Mice lacking B-cells, however, show no protracted illness and clear the parasite just as well as wild-type mice Sateriale et al.

Genetic modification of the C. Originally pioneered in C. For those genes that are found to be essential, or for the temporal control of protein expression, the newly developed technique of conditional protein degradation in Cryptosporidium should prove very useful Choudhary et al.

With C. The most widely used cell culture model for Cryptosporidium infection is the ileocecal adenocarcinoma cell line HCT-8 Upton et al. This cell line has served as the primary infection model for anti-cryptosporidial drug screening and has provided many important insights into the biology of infection Love et al.

However, HCT-8s and the standard 2-D model of infection is restrictive in two crucial aspects: 1 Cell monolayers lack the cell diversity and architecture of the host intestine that is important for investigating pathogenesis, and 2 parasite growth is short-lived in this system as they are unable to complete the sexual stage of their life cycle.

Recent research has implicated this sexual cycle, specifically the inability of male parasite gametes to fertilize female gametes, as the restrictive stage in the 2-D cell monolayer infection model Tandel et al. This breakdown of fertilization is hypothesized to cause the arrested parasite growth that occurs around 72 hours after infection of HCT-8s and other 2-D epithelial cell models.

However, several in vitro systems have been able to sustain long-lived Cryptosporidium infections by recapitulating conditions that more closely mimic the host intestine.

The developing organoid model system of in vitro cell culture has been a game-changer for stem cell and cancer biologists, with infectious disease studies following closely behind [for a comprehensive review see Rossi et al. In brief, organoids are three-dimensional growths arising from embryonic, induced pluripotent, or adult stem cells. When given proper differentiation signals and an extracellular matrix ECM -derived scaffold, stem cells can develop into avatars of the desired organ.

Organoids from adult stem cells recapitulate most of the architecture and cell types characteristic of the tissue from which they were derived and hence can be used to study a range of aspects including basic cell and tumor biology, organ regeneration, and increasingly host-pathogen interactions in a more physiologically relevant setting.

Organoids of many different systems including the brain, liver, and lung have been successfully created; however, arguably the most well-established and studied platform is that of the intestinal organoid, which has been well-characterized from both mice and men Sato et al. In a mixture of ECM proteins, small intestinal crypt-derived stem cells self-organize into mini-organs consisting of differentiated enterocytes and secretory cells, while still maintaining intestinal stem cells, a strict apical-basal polarity, and basic crypt-villus architecture Figure 2B Sato et al.

Recently, Heo et al. Transmission electron microscopy TEM showed the presence of asexual parasite stages 1 day post-infection, as well as the presence of sexual stages and oocysts after 5 days. TEM of infected organoids was also able to indicate that enterocytes in SI organoids, but both secretory and non-secretory cells in lung organoids, were predominantly infected by Cryptosporidium. New oocysts produced in organoids were capable of maintaining productive infections over three rounds of serial passage in SI organoids, however the authors noted a reduction in parasite numbers over 4 weeks of passaging.

High-throughput RNAseq analysis of infected organoids revealed the activation of an enterocyte-based interferon response. Analysis of parasite RNA expression also demonstrated the upregulation of oocyst wall genes especially COWP1 at 72 hours in both SI and lung organoids, a finding that supports the observation of new oocyst generation in this system. It should be noted, however, that female gametes produce oocyst wall proteins even in the absence of fertilization from male gametes Tandel et al.

Male or microgamete specific genes such as HAP2 were not identified as being upregulated in this study. Interestingly, C. Originally devised for the culture of primary nasal epithelial cells, another recently-developed system to create differentiated intestinal cells from stem cells is the air-liquid interface ALI culture Figure 2C Wang et al.

Here, stem cells either from primary sources or previously maintained as spheroids can be plated onto 2-D membrane-supported cell culture inserts with or without an ECM-derived gel base. A layer of irradiated fibroblasts can also be used as feeder cells. Removal of liquid media from the top of the inserts results in the differentiation of stem cells into enterocyte and secretory cells.

This technique was recently adapted by Wilke et al. Parasite growth monitored by quantitative PCR showed amplification by nearly fold over 20 days. Similar to the organoid model, ALI cultures were capable of producing new infectious oocysts 3 days post-infection; oocysts were capable of infecting both mice and new membrane-supported cell monolayers, albeit with diminishing returns. Also similar to the organoid model, TEM showed the preferential infection of enterocytes, however some infected secretory cells were also reported.

Interestingly again, C. This meiotic recombination event, which naturally occurs in the final stages of the Cryptosporidium life cycle, appears in their ALI model after the third day of infection and coincides with the production of new infectious oocysts.

The ability to perform controlled genetic crosses of Cryptosporidium parasites using a cell culture platform is a major advancement for the field. Exploring the nature and frequency of recombination events and how they contribute to pathogenicity and ongoing evolution of Cryptosporidium will greatly further the study of this deadly parasite.

Children living in endemic regions are particularly vulnerable to Cryptosporidium infection and this is well evidenced in large scale studies of diarrheal aetiology.

In the Global Enteric Multicenter GEM study, with a cohort of over 20, children across Africa and southeast Asia, Cryptosporidium was identified as one of the primary causes of severe diarrheal events Kotloff et al. However, from the third year onward, cryptosporidiosis becomes a negligible infection with very few attributable cases of severe disease reported in the GEM study.

Early results using C. Mice that recover from a natural C. Vaccination using irradiated parasites had the same effect of protecting against subsequent infection and disease. Unlike its relative Plasmodium , Cryptosporidium appears to lack the large gene families required for antigenic switching Abrahamsen et al. This may be a great advantage for development of a vaccine, yet researchers first need to understand the mechanism and markers of Cryptosporidium immunity.

The true power of this newly developed mouse model of cryptosporidiosis lies in the ability to genetically manipulate both the host and parasite. There is a vast range of mouse species, strains, congenics and transgenics to explore pathogenesis. Next generation panels of recombinant inbred lines, such as the Collaborative Cross, can be leveraged for genome-wide interrogation of the host response to Cryptosporidium infections Aylor et al.

This new mouse model of infection holds the potential to study both innate and adaptive mechanisms of immunity and, coupled with stem cell-based in vitro culture, microscopic observations can be studied in controlled environments. Both the organoid and ALI culture systems raise the possibility of conducting in vitro studies of immune cell interaction with Cryptosporidium -infected epithelial cells.



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With the pandemic affecting economic conditions, demand for smartphones is sluggish and consumers are waiting longer to upgrade, but as the world's fastest growing smartphone company, Realme can buck that trend, Li added. Data from consultancy IDC shows the European market was comparable to the United States in the first three quarters of in terms of smartphone revenues, but lagged behind China. Spun off from fellow Chinese smartphone maker Oppo in , Realme was the world's sixth-biggest smartphone seller as of end-September, according to Counterpoint, with strong sales in India, Southeast Asia and Eastern Europe. Li said he expects the G2 Pro phone to appeal to European customers because it is one of the first phones to launch with Qualcomm's new flagship Snapdragon 8gen1 processor, which promises higher speed and power efficiency. Realme sold 60 million handsets in globally and aims to sell 90 million this year and over million in , Li said. The company, like its peers, has been hit by semiconductor shortages over the last year, but Li said the constraints could ease by the second half of


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