Human Intestinal Epithelial Cells Respond to Cryptosporidium parvum Infection with Increased Prostaglandin H Synthase 2 Expression and Prostaglandin E2 and F2α Production

Cryptosporidium parvum is an important cause of diarrhea in humans and several animal species. Prostaglandins play a central role in regulating intestinal fluid secretion in animal models of cryptosporidiosis, but their cellular sources and mechanisms of induction are unclear. Here, we show that C. parvum infection directly activates prostaglandin H synthase 2 expression and prostaglandin E₂ and F_2α production in human intestinal epithelial cells.     

Infection of immunocompetent mice with acid-water-pretreated Cryptosporidium parvum results in weight loss, and intestinal (structural and physiological) alterations

Cryptosporidiosis, caused by Cryptosporidium, causes self-limited diarrhea in normal hosts but may cause life-threatening diarrhea in immunocompromised persons. Cryptosporidium-induced manifestations, including weight-loss and intestinal physiological alterations are not noted in adult immunocompetent mice. So far, studies that have been used to test the therapeutic efficacy of drugs have been performed using various immunocompromised animal models. There is an urgent need of an immunocompetent small animal model that portrays Cryptosporidium-induced manifestations. In the current studies, we have compared two Cryptosporidium parvum pretreatment methods, we have hence used sodium hypochlorite or acidic water to treat Cryptosporidium parvum, followed by infection by oral gavage in adult immunocompetent C57BL6 mice. We demonstrated manifestations such as weight loss, intestinal structural and physiological alterations such as intestinal, villi blunting, and glucose malabsorption (as studied by the Ussing chamber technique) only in response to infection with C. parvum that has been treated with acidic water and not with sodium hypochlorite. These novel studies reveal that acidic water treatment of C. parvum results in manifestations of cryptosporidiosis in otherwise resistant immunocompetent mice. The current studies open up possibilities of using the normal immunocompetent mice model to test therapeutic drugs against cryptosporidiosis. An erratum to this article can be found at     

Inhibition of apoptosis in Cryptosporidium parvum-infected intestinal epithelial cells is dependent on survivin

Cryptosporidium parvum is an obligate intracellular protozoan capable of causing severe diarrheal disease in a wide variety of mammals, including humans. C. parvum infection has been associated with induction of apoptosis in exposed epithelial cells, and we now demonstrate that apoptosis is restricted to a subset of cells actively infected with C. parvum. Approximately 20% of the infected cells underwent apoptosis within 48 h of infection, suggesting that the majority of the infected cells are rescued from apoptosis. C. parvum infection resulted in low-level activation of multiple members of the caspase family, including caspase-2, -3, -4, -6, -8, and -9. The kinetics of caspase activation correlated with apoptosis over a 48-h time course. Pan caspase inhibitors reduced apoptosis of epithelial cells infected by C. parvum. Furthermore, C. parvum infection inhibited staurosporine-induced apoptosis and caspase-3/7 activation at 24 h and 48 h. Infection with C. parvum led to upregulation of genes encoding inhibitors of apoptosis proteins (IAPs), including c-IAP1, c-IAP2, XIAP, and survivin. Knockdown of survivin gene expression, but not that of c-IAP1, c-IAP2, or XIAP expression, increased caspase-3/7 activity as well as apoptosis of infected cells and decreased C. parvum 18S rRNA levels. These data suggest that the apoptotic response of infected intestinal epithelial cells is actively suppressed by C. parvum via upregulation of survivin, favoring parasite infection.     

Role of Immunoglobulin A Monoclonal Antibodies against P23 in Controlling Murine Cryptosporidium parvum Infection

Cryptosporidium parvum is an important diarrhea-causing protozoan parasite of immunocompetent and immunocompromised hosts. Immunoglobulin A (IgA) has been implicated in resistance to mucosal infections with bacteria, viruses, and parasites, but little is known about the role of IgA in the control of C. parvum infection. We assessed the role of IgA during C. parvum infection in neonatal mice. IgA-secreting hybridomas were developed by using Peyer’s patch lymphocytes from BALB/c mice which had been orally inoculated with viable C. parvum oocysts. Six monoclonal antibodies (MAbs) were selected for further study based on indirect immunofluorescence assay reactivity with sporozoite and merozoite pellicles and the antigen (Ag) deposited on glass substrate by gliding sporozoites. Each MAb was secreted in dimeric form and recognized a 23-kDa sporozoite Ag in Western immunoblots. The Ag recognized comigrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with P23, a previously defined neutralization-sensitive zoite pellicle Ag. MAbs were evaluated for prophylactic or therapeutic efficacy against C. parvum, singly and in combinations, in neonatal BALB/c mice. A combination of two MAbs given prophylactically prior to and 12 h following oocyst challenge reduced the number of intestinal parasites scored histologically by 21.1% compared to the numbers in mice given an isotype-matched control MAb (P < 0.01). Individual MAbs given therapeutically in nine doses over a 96-h period following oocyst challenge increased efficacy against C. parvum infection. Four MAbs given therapeutically each reduced intestinal infection 34.4 to 42.2% compared to isotype-matched control MAb-treated mice (P < 0.05). One MAb reduced infection 63.3 and 72.7% in replicate experiments compared to isotype-matched control MAb-treated mice (P < 0.0001). We conclude that IgA MAbs directed to neutralization-sensitive P23 epitopes may have utility in passive immunization against murine C. parvum infection.     

Cell phenotypic change due to Cryptosporidium parvum infection in immunocompetent mice

Cryptosporidium parvum is an intracellular parasite causing enteritis which can become life-threatening in immunocompromised host. Immunoregulatory T cells play a central role in the regulatory network of the host. Here, we proposed to characterize the populations of immune cells during infection and reinfection with C. parvum. Four-week-old BALB/C mice were inoculated with oocysts of C. parvum at days 0 and 22. Fecal and blood samples, spleens, and small intestines were collected for analysis. Peripheral blood and spleen cell populations were characterized by flow cytometry. After infection (days 0 to 21), mice presented higher values of neutrophils, eosinophils, NK cells and CD4⁺CD25^high T cells in peripheral blood. After reinfection, this upward trend continued in the following days for all four populations in infected mice. At day 35, infected mice presented similar values to the control group, except for CD4⁺CD25^high T cells, which remained higher in infected mice. A possible correlation between alterations in blood and spleen cell populations was also studied, but no consistent association could be established. Small intestine sections were screened for intracellular stages of the parasite but no evidence of pathology was observed. Here, we report information which may be important for the understanding of the specific cell-mediated response in immunocompetent mice to C. parvum infection. Although some questions remain unanswered and complementary studies are needed, our results are expected to contribute to a better understanding of innate and Treg cells role in the clearance process of this parasite.     

Subgenotype analysis of Cryptosporidium parvum isolates from humans and animals in Japan using the 60-kDa glycoprotein gene sequences

Cryptosporidium parvum is a well-known intestinal parasite which is associated with severe acute diarrhea in humans and animals. This parasite is composed of morphologically identical but genetically different multiple genotypes. In humans, cryptosporidiosis is mainly caused by two C. parvum genotypes, human genotype (previously known as genotype 1 and recently proposed as new species C. hominis) and cattle genotype (previously known as genotype 2). However, recent molecular studies indicate the genetic heterogeneity among the isolates of C. parvum human or cattle genotype. Therefore, identification of the isolates at the subgenotype level is more useful for control of the Cryptosporidium infection or for understanding of the population structure of C. parvum genotypes. In the present study, we identified the subgenotypes of the C. parvum human or cattle genotype isolates from humans and animals in Japan using DNA sequencing analysis of the C. parvum 60-kDa glycoprotein gene (GP60) and showed the new subgenotype in a raccoon dog isolate. This study suggested that C. parvum cattle genotype might be composed of zoonotic and host-specific multiple subgenotypes.     

<Emphasis Type="Italic">Cryptosporidium parvum</Emphasis> infection stimulates the secretion of TGF-β, IL-8 and RANTES by Caco-2 cell line

Cryptosporidium parvum is a common cause of diarrhea in humans. Although mild inflammatory mucosal infiltrate is usually observed, limited information is currently available on the pathogenic mechanisms involved in this phenomenon. The aim of this work was to investigate in vitro the influence of C. parvum infection on the secretion of lymphocyte-targeted chemokines (RANTES, MIP-1α, MIP-1β, IL-8), proinflammatory cytokines (TNF-α, GM-CSF and IL-6) and TGF-β by human enterocytic Caco-2 cells. C. parvum infection stimulates IL-8, RANTES and TGF-β secretion by both the basal and apical side of caco-2 cells. A slight increase in TNF-α production by infected cells was observed in the apical compartment. Data suggest that enterocytic chemokines and/or TGF-β are involved in the initiation and regulation of the mucosal response to C. parvum infection.     

Requirement for CD40-CD40 Ligand Interaction for Elimination of Cryptosporidium parvum from Mice

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.     

Human Cryptosporidiosis Caused by Cryptosporidium tyzzeri and C. parvum Isolates Presumably Transmitted from Wild Mice

We report a case of severe human cryptosporidiosis caused by Cryptosporidium tyzzeri and C. parvum with an unusually high frequency of liquid stools. Wild mice were the most likely source of infection, demonstrating the potential for wild-mouse-borne Cryptosporidium to infect humans and highlighting the health risks associated with synantropic rodents.     

Susceptibility and Serologic Response of Healthy Adults to Reinfection with Cryptosporidium parvum

Healthy adults are susceptible to infection with small numbers of Cryptosporidium parvum oocysts, resulting in self-limited infection. We investigated if infection of humans with C. parvum is protective 1 year after primary exposure. At 1 year after a primary challenge with 30 to 10⁶ oocysts, 19 healthy immunocompetent adults were rechallenged with 500 oocysts and monitored for the development of infection and/or illness. Oocyst excretion was quantitated by direct immunofluorescence with a C. parvum-specific monoclonal antibody, and anti-C. parvum antibodies in serum were detected by an enzyme-linked immunosorbent assay. Fewer subjects shed oocysts after the second exposure (3 of 19; 16%) than after the first exposure (12 of 19; 63%) (P < 0.005). Although the rates of diarrhea were comparable after each of the two exposures, the clinical severity as determined by the mean number of unformed stools passed was lower after reexposure (11.25 versus 8.62; P < 0.05). The number of anti-Cryptosporidium immunoglobulin G and A seroconversions increased after secondary exposure. However, the C. parvum serum antibody response did not correlate with the presence or absence of infection.     

Cryptosporidium parvum induces SIRT1 expression in host epithelial cells through downregulating let-7i

Epithelial cells along human gastrointestinal mucosal surface express pathogen-recognizing receptors and actively participate in the regulation of inflammatory reactions in response to microbial infection. The NAD-dependent deacetylase sirtuin-1 (SIRT1), one member of the sirtuin family of proteins and an NAD-dependent deacetylase has been implicated in the regulation of multiple cellular processes, including inflammation, longevity, and metabolism. In this study, we demonstrated that infection of cultured human biliary epithelial cells (H69 cholangiocytes) with a parasitic protozoan, Cryptosporidium parvum, induced SIRT1 expression at the protein level without a change in SIRT1 mRNA content. Using real-time PCR and Northern blot analyses, we found that C. parvum infection decreased the expression of let-7i in infected H69 cells. Down-regulation of let-7i caused relief of miRNA-mediated translational suppression of SIRT1 and consequently, resulted in an increased SIRT1 protein level in infected H69 cell cultures. Moreover, gain- and loss-of-function studies revealed that let-7i could modulate NF-κB activation through modification of SIRT1 protein expression. Thus, our data suggest that let-7i regulates SIRT1 expression in human biliary epithelial cells in response to microbial challenge, suggesting a new role of let-7i in the regulation of NF-κB-mediated epithelial innate immune response.     

Contributions of NanI Sialidase to Caco-2 Cell Adherence by Clostridium perfringens Type A and C Strains Causing Human Intestinal Disease

Previous studies showed that Clostridium perfringens type D animal disease strain CN3718 uses NanI sialidase for adhering to enterocyte-like Caco-2 cells. The current study analyzed whether NanI is similarly important when type A and C human intestinal disease strains attach to Caco-2 cells. A PCR survey determined that the nanI gene was absent from typical type A food poisoning (FP) strains carrying a chromosomal enterotoxin (CPE) gene or the genetically related type C Darmbrand (Db) strains. However, the nanI gene was present in type A strains from healthy humans, type A strains causing CPE-associated antibiotic-associated diarrhea (AAD) or sporadic diarrhea (SD), and type C Pig-Bel strains. Consistent with NanI sialidase being the major C. perfringens sialidase when produced, FP and Db strains had little supernatant sialidase activity compared to other type A or C human intestinal strains. All type A and C human intestinal strains bound to Caco-2 cells, but NanI-producing strains had higher attachment levels. When produced, NanI can contribute to host cell attachment of human intestinal disease strains, since a nanI null mutant constructed in type A SD strain F4969 had lower Caco-2 cell adhesion than wild-type F4969 or a complemented strain. Further supporting a role for NanI in host cell attachment, sialidase inhibitors reduced F4969 adhesion to Caco-2 cells. Collectively, these results suggest that NanI may contribute to the intestinal attachment and colonization needed for the chronic diarrhea of CPE-associated AAD and SD, but this sialidase appears to be dispensable for the acute pathogenesis of type A FP or type C enteritis necroticans.     

Roles for NK Cells and an NK Cell-Independent Source of Intestinal Gamma Interferon for Innate Immunity to Cryptosporidium parvum Infection

A gamma interferon (IFN-γ)-dependent innate immune response operates against the intestinal parasite Cryptosporidium parvum in T- and B-cell-deficient SCID mice. Although NK cells are a major source of IFN-γ in innate immunity, their protective role against C. parvum has been unclear. The role of NK cells in innate immunity was investigated using Rag2^−/− mice, which lack T and B cells, and Rag2^−/− γ_c^−/− mice, which, in addition, lack NK cells. Adult mice of both knockout lines developed progressive chronic infections; however, on most days the level of oocyst excretion was higher in Rag2^−/− γ_c^−/− mice and these animals developed morbidity and died, whereas within the same period the Rag2^−/− mice appeared healthy. Neonatal mice of both mouse lines survived a rapid onset of infection that reached a higher intensity in Rag2^−/− γ_c^−/− mice. Significantly, similar levels of intestinal IFN-γ mRNA were expressed in Rag2^−/− and Rag2^−/− γ_c^−/− mice. Also, infections in each mouse line were exacerbated by treatment with anti-IFN-γ neutralizing antibodies. These results support a protective role for NK cells and IFN-γ in innate immunity against C. parvum. In addition, the study implies that an intestinal cell type other than NK cells may be an important source of IFN-γ during infection and that NK cells may have an IFN-γ-independent protective role.Cryptosporidiosis is an infectious diarrheal disease that affects different types of vertebrates, including mammals (3). The etiological agent is the monoxenous protozoan parasite Cryptosporidium, which belongs to the Apicomplexa. One species, Cryptosporidium hominis, may have a predilection for infecting humans, while a morphologically similar parasite, Cryptosporidium parvum, readily infects both cattle and humans (3). The cryptosporidia of mammals invade intestinal epithelial cells, where they multiply asexually to produce merozoites that infect more cells. Eventually, merozoites may undergo differentiation into gamonts that form new oocysts, containing four sporozoites, and the oocysts transmit infection to new hosts by the fecal-oral route. The clinical phase of cryptosporidiosis normally lasts a few days but may persist and become fatal in immunocompromised hosts (2).Studies of protective host immune responses to Cryptosporidium indicate that elimination of infection involves adaptive immunity and, in particular, requires the presence of CD4⁺ T cells. AIDS patients with low CD4⁺ cell counts have shown increased susceptibility to cryptosporidial infection and high rates of morbidity and mortality, while resolution of AIDS-associated infection following anti-human-immunodeficiency-virus drug treatment coincided with the partial recovery of intestinal CD4⁺ T-cell counts (2, 23). Mice with a CD4⁺ T-cell deficiency were found to be incapable of clearing C. parvum infection (1), and similarly, depletion of these cells from immunocompetent animals with specific antibody increased oocyst production (27). CD4⁺ T cells are also an important source of gamma interferon (IFN-γ), and this cytokine plays a key role in the control of infection. Antigen-specific IFN-γ production by restimulated CD4⁺ T cells from humans who recovered from infection was observed, although cells taken during acute infection were not responsive to antigen (6). IFN-γ^−/− mice or mice administered anti-IFN-γ neutralizing antibodies had exacerbated infections compared with control animals (18, 27). IFN-γ activity during C. parvum infection has been associated with a chemokine response by intestinal epithelial cells that attracted both CD4⁺ T cells and macrophages into the lamina propria (10). In addition, IFN-γ has been shown to have a direct effect on parasite growth by activating epithelial cell antimicrobial killing activity (19).Innate immune responses are also able to limit the reproduction of C. parvum. Immunocompromised adult nude mice (lacking T cells) or SCID mice (lacking T and B cells) developed chronic infections that were controlled for a number of weeks but eventually became progressive and fatal (13, 17, 27). IFN-γ was important for the initial resistance of these mice, since administration of anti-IFN-γ neutralizing antibodies to adult or neonatal SCID mice increased susceptibility to infection (14, 28), and repeated antibody treatment resulted in rapid establishment of severe infection (14). In addition, morbidity as a result of parasite reproduction appeared sooner in SCID IFN-γ^−/− mice than in SCID mice (7).NK cells are involved in resistance to intracellular microbial pathogens, including protozoa, and are a major source of IFN-γ in innate immunity (9). NK cells originate mainly in the bone marrow, from where they migrate to other organs (5, 29). Interleukin-15 (IL-15) is essential for differentiation and subsequent survival of NK cells and can also be important in activation of the cells (5, 9). NK cells are activated by ancillary cells, such as dendritic cells (DCs), by direct contact and by proinflammatory cytokines produced by DCs stimulated by antigen (9). Activated NK cells produce IFN-γ and other proinflammatory cytokines and may also become cytotoxic against infected cells.The protective role of NK cells in innate immunity to C. parvum is unclear, but some studies imply that these cells may be involved. Human peripheral blood NK cells treated with IL-15 were shown to have cytolytic activity against human intestinal epithelial cell lines infected with C. parvum (4), and intestinal expression of this cytokine has been detected in humans (20). C. parvum infection was found to be more widespread in SCID mice deficient in NK cell cytotoxicity than in SCID mice with normal NK cell function (17). In addition, in vitro studies demonstrated that splenocytes from SCID mice produced IFN-γ in the presence of cryptosporidial antigens, but if NK cells were depleted, IFN-γ production did not occur (15). However, attempts to show that NK cells were protective in SCID mice infected with C. parvum have not been successful. In separate studies, treatment of these mice with anti-asialo-GM1 antibodies that can deplete NK cells in vivo was shown to have no effect on the course of C. parvum infection (15, 27), and while it has been argued that these antibodies might not have reached the gut in sufficient quantity to be effective, similar antibodies were shown to diminish intestinal NK cell function (30).The aim of the present study was to examine further the role of NK cells and IFN-γ in the innate immune response to C. parvum. The pattern of infection and immune responses were compared in Rag2^−/− mice, which lack T and B cells, and Rag2^−/− γ_c^−/− mice, which, in addition, lack NK cells due to the absence of the γ_c chain component of the IL-15 receptor (5). The results support protective roles for IFN-γ and NK cells in innate immunity to C. parvum but also indicate that IFN-γ from a cell type other than NK cells is important for control of infection.     

Attachment of Cryptosporidium parvum Sporozoites to Human Intestinal Epithelial Cells

An enzyme-linked immunosorbent assay-based attachment model using the human intestinal cell line Caco-2A was developed to study attachment of Cryptosporidium parvum sporozoites in vitro and to assess potential inhibitors of sporozoite binding. In this system, attachment was related to sporozoite dose, incubation time, and host cell differentiation status. Polyclonal antibodies to C. parvum as well as glycoprotein inhibitors of a sporozoite lectin reduced attachment. This model will be a valuable tool in elucidating specific molecules and mechanisms involved in sporozoite-host cell attachment.     

Oral Immunization with Attenuated Salmonella enterica Serovar Typhimurium Encoding Cryptosporidium parvum Cp23 and Cp40 Antigens Induces a Specific Immune Response in Mice

Attenuated Salmonella enterica serovar Typhimurium vaccine strain SL3261 was used as an antigen delivery system for the oral immunization of mice against two Cryptosporidium parvum antigens, Cp23 and Cp40. Each antigen was subcloned into the pTECH1 vector system, which allows them to be expressed as fusion proteins with highly immunogenic fragment C of tetanus toxin under the control of the anaerobically inducible nirB promoter. The recombinant vector was introduced into Salmonella Typhimurium vaccine strain SL3261, and the stable soluble expression of the chimeric protein was evaluated and confirmed by Western blotting with polyclonal C. parvum antisera. Mice were inoculated orally with a single dose of SL3261/pTECH-Cp23 or Cp40, respectively, and plasmid stability was demonstrated both in vitro and in vivo. Specific serum immunoglobulin G (IgG) antibodies against the Cp23 or Cp40 antigen were detected by enzyme-linked immunosorbent assay 35 days after immunization. Also, serum IgA and mucosal (feces) IgA antibodies were detected in 30% of the mice immunized with Cp23. In addition, prime-boosting with Cp23 and Cp40 DNA vaccine vectors followed by Salmonella immunization significantly increased antibody responses to both antigens. Our data show that a single oral inoculation with recombinant S. Typhimurium SL3261 can induce specific antibody responses to the Cp23 or Cp40 antigen from C. parvum in mice, suggesting that recombinant Salmonella is a feasible delivery system for a vaccine against C. parvum infection.Cryptosporidium parvum is an obligate intracellular parasite that infects intestinal epithelial cells and has been identified as being a significant cause of diarrheal disease in a variety of mammalian species including rodents, livestock, and humans (24). Infection is usually self-limiting in immunocompetent individuals but can be severe and even life-threatening for those that have compromised immune systems, such as human immunodeficiency virus-infected individuals, transplant recipients, children, and the elderly (30). The incidence of cryptosporidiosis has been reported to be in the range of 1 to 10% (34) but has been reported to be as high as 30% in children in India and Saudi Arabia (1, 10, 14). In light of the fact that chemotherapeutic agents for the treatment of infections of immunodeficient individuals are limited and not always efficacious, the development of a vaccine that is capable of inducing at least partial protection would be beneficial to specific high-risk populations. Data from human volunteer studies have suggested that at least partial immunity develops, as subsequent exposures with the parasite resulted in less-severe clinical signs (26).Since all life cycle stages occur in the host epithelium, the mucosal immune response is paramount to providing resistance and protection. The use of live oral Salmonella vaccines has been successful at delivering heterologous antigens and at generating a mucosal immune response against a number of organisms including intestinal parasitic species such as Toxoplasma gondii and Eimeria tenella (18, 29). Advantages of attenuated Salmonella vaccines include the fact that they induce both cell-mediated and humoral responses, elicit a systemic and local response, are easy to administer, and are affordable (13). To date, reports of the use of attenuated Salmonella as a vaccine vector in C. parvum are not available. Through this study, we assessed the use of an attenuated Salmonella strain carrying specific C. parvum antigens as a vaccine vector and the potential that it offers against C. parvum infection.In this study, we compared the abilities of attenuated strains of Salmonella to express the immunodominant antigens Cp23 and Cp40. These surface antigens of C. parvum are considered to be immunodominant since they are recognized by serum antibodies of humans and many other animals (25, 31, 36). Moreover, the level of oocyst secretion was reduced following the administration of colostrum directed against the Cp23 antigen (26). T-cell responses to Cp23 from infected mice (3), calves (36), and human peripheral blood mononuclear cells (33) with C. parvum infection have been reported, indicating its role in the immune response to C. parvum. Recombinant Cp40 antigen was previously shown to generate a T-cell proliferation response in mice (31). Also, monoclonal antibodies against Cp40 antigen have been shown to neutralize C. parvum infection and inhibit attachment in vitro (4). We also report the safety and plasmid stability of the Salmonella vaccine vector in mice as well as the ability to induce an antibody response against the expressed antigens.     

Cryptosporidium parvum Initiates Inflammatory Bowel Disease in Germfree T Cell Receptor-α-Deficient Mice

Flora-bearing mice with targeted disruption of T cell receptor (TCR)-α or -β genes spontaneously develop intestinal inflammation with features similar to ulcerative colitis in humans. TCR-α-deficient mice maintained germfree or colonized with a limited number of intestinal bacteria failed to develop inflammatory bowel disease (IBD)-like lesions. Evidently, inflammation in these mice does not develop spontaneously or result from a generalized antigenic stimulation, but rather requires induction by a heretofore unidentified specific stimulus. We describe the development of IBD-like lesions in germfree TCR-α-deficient mice monoassociated with the protozoan Cryptosporidium parvum. Lesions were seen in distal ileum, cecum, and colon and were most severe in the cecum. A prominent leukocytic infiltrate within the lamina propria was a common characteristic of the lesions observed in the C. parvum-infected germfree TCR-α-deficient mice. The leukocytic infiltrate was composed of aggregates of B220⁺ cells, the majority of which expressed surface IgD (ie, conventional B lymphocytes). It has been proposed that antigenic stimulation by a microorganism(s) is needed to initiate intestinal inflammation in TCR-α-deficient mice. Our results indicate that a single microbial species, C. parvum, is capable of triggering the development of IBD-like lesions in germfree TCR-α-deficient mice.     

Lymphocytes and not IFN-γ mediate expression of iNOS by intestinal epithelium in murine cryptosporidiosis

We hypothesized that unrecognized differences in epithelial expression of inducible nitric oxide synthase (iNOS), resulting from engineered immunodeficiency, could explain the contradictory findings of prior studies regarding the importance of nitric oxide (NO) in murine models of Cryptosporidium parvum infection. Severe combined immunodeficient mice (SCID) failed to constitutively or inducibly express epithelial iNOS or increase NO synthesis in response to C. parvum infection. In contrast, mice lacking IFN-γ alone induced both epithelial iNOS expression and NO synthesis in response to infection. Accordingly, lymphocytes mediate epithelial expression of iNOS and NO synthesis independent of IFN-γ in response to C. parvum infection. These findings in large part explain the contradictory conclusions of prior studies regarding the role of iNOS in C. parvum infection.     

Kinetics of Cryptosporidium parvum-specific cytokine responses in healing and nonhealing murine models of C. parvum infection

Susceptibility or resistance to infection with Cryptosporidium parvum correlates with the ability of mice to produce characteristic panels of cytokines in response to infection. Both adult healing and nonhealing mouse models of cryptosporidiosis were used to study the cell-mediated immune response during the course of C. parvum infection. Mesenteric lymph node (MLN) lymphocytes from both mouse models were proliferated after ex vivo re-stimulation with C. parvum sporozoite antigen. Study of the cytokine profile from the supernatant of proliferated MLN cells revealed that healing mice produced greater levels of Th1 (IFN- and IL-2) and moderate amounts of Th2 (IL-4, IL-5, IL-6, and IL-10) cytokines throughout the course of infection. Whereas, MLN cells from nonhealing mice produced no IFN-, low levels of IL-2 and IL-4, and higher levels of IL-5, IL-6, and IL-10 cytokines. These results suggest that the capacity to produce both Th1 and Th2 cytokines, rather than the presence of Th2 cytokines alone, determines the effective immune response against C. parvum infection.     

Persistence and Toxin Production by Clostridium difficile within Human Intestinal Organoids Result in Disruption of Epithelial Paracellular Barrier Function

Clostridium difficile is the leading cause of infectious nosocomial diarrhea. The pathogenesis of C. difficile infection (CDI) results from the interactions between the pathogen, intestinal epithelium, host immune system, and gastrointestinal microbiota. Previous studies of the host-pathogen interaction in CDI have utilized either simple cell monolayers or in vivo models. While much has been learned by utilizing these approaches, little is known about the direct interaction of the bacterium with a complex host epithelium. Here, we asked if human intestinal organoids (HIOs), which are derived from pluripotent stem cells and demonstrate small intestinal morphology and physiology, could be used to study the pathogenesis of the obligate anaerobe C. difficile. Vegetative C. difficile, microinjected into the lumen of HIOs, persisted in a viable state for up to 12 h. Upon colonization with C. difficile VPI 10463, the HIO epithelium is markedly disrupted, resulting in the loss of paracellular barrier function. Since similar effects were not observed when HIOs were colonized with the nontoxigenic C. difficile strain F200, we directly tested the role of toxin using TcdA and TcdB purified from VPI 10463. We show that the injection of TcdA replicates the disruption of the epithelial barrier function and structure observed in HIOs colonized with viable C. difficile.