Shannon N. Moonah

Burden of disease from cryptosporidiosis.

Purpose of review The global significance of cryptosporidiosis is widespread and far-reaching. In this review, we present recent data about strain diversity and the burden of disease, along with developments in therapeutic and preventive strategies. Recent findings Cryptosporidium is an emerging pathogen that disproportionately affects children in developing countries and immunocompromised individuals. Without a diagnostic tool amenable for use in developing countries, the burden of infection and its relationship to growth faltering, malnutrition, and diarrheal mortality remain underappreciated. Disease incidence is also increasing in industrialized countries largely as a result of outbreaks in recreational water facilities. Advances in molecular methods, including subtyping analysis, have yielded new insights into the epidemiology of cryptosporidiosis. However, without practical point-of-care diagnostics, an effective treatment for immunocompromised patients, and a promising vaccine candidate, the ability to reduce the burden of disease in the near future is limited. This is compounded by inadequate coverage with antiretroviral therapy in developing countries, the only current means of managing HIV-infected patients with cryptosporidiosis. Summary Cryptosporidiosis is one of the most important diarrheal pathogens affecting people worldwide. Effective methods to control and treat cryptosporidiosis among high-risk groups present an ongoing problem in need of attention.

Host Immune Response to Intestinal Amebiasis

Entamoeba histolytica is an invasive enteric protozoan parasite that causes amebiasis. Globally, diarrheal disease is second only to pneumonia as a leading cause of death in children under five, and intestinal amebiasis is one of the top ten causes of severe diarrhea in the developing world. Amebiasis is more common in malnourished children, a state that afflicts approximately one-third of children in the developing world. In the critical first year of life, 11% of Bangladeshi infants living in poverty suffer from E. histolytica diarrhea [1], [2]. There is currently no vaccine for this devastating disease, thus an understanding of the human immune response toward the parasite would greatly enhance the ability to develop effective immunotherapies. The host deploys a series of immune defenses against the parasite as it invades the colon. The ameba, however, has developed complex strategies to evade host defenses and promote its own survival. Here, we summarize the dynamics of the interaction of parasite with host and its importance in the pathogenesis of amebiasis (Figure 1). Figure 1 A. Host Immune Response to Intestinal Amebiasis. Innate Immunity Stomach acid serves as an important first line of defense against enteropathogens through its ability to kill acid-sensitive microorganisms. However, infectious amebic cysts are highly resistant and survive passage through the acidic environment of the stomach. In the intestine, the next layer of innate defense may be the mucus layer, which is thought to act as a protective barrier, preventing E. histolytica from invading intestinal epithelial cells (IECs). Mucin, a major constituent of the intestinal mucus layer, is a glycoprotein secreted by goblet cells and submucosal glands. Mucin glycoproteins bind to and inhibit the Gal/GalNAc adherence lectin of the parasite, preventing in vitro adherence and killing of CHO cells [3]. Trophozoites, however, can disrupt the mucus layer and intestinal barrier by secreting cysteine proteases (CPs) and glycosidases to allow for penetration of the colonic mucosa. Specifically, E. histolytica cysteine protease-A5 (EhCP-A5) degrades mucin-2 (MUC2) and extracellular matrix (ECM) proteins [4]. The importance of cysteine proteases was demonstrated by an ex vivo human intestinal model, where EhCP-A5–silenced parasites failed to penetrate into the colonic lamina propria [5]. IECs exposed to E. histolytica trophozoites secrete potent chemokines, such as IL-8, resulting in immune cell recruitment and infiltration of the lamina propria and intestinal epithelium [6]. Neutrophils are one of the first immune cells to respond to amebic invasion. Neutrophils activated by interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), or lipopolysaccharides (LPS) carry out amebicidal activity in vitro by releasing reactive oxygen species (ROS) [7], [8]. Depletion of neutrophils with anti-Gr-1 antibodies resulted in exacerbated intestinal disease in murine models, supporting the protective role of neutrophils in amebiasis [9]. It should be noted, however, that anti-Gr-1 antibodies can deplete other granulocytes such as eosinophils. Macrophages also play a crucial role in the host response against intestinal amebiasis. Macrophages are amebicidal after stimulation with IFN-γ or TNF-α [10], [11]. Several amebic antigens are known to activate these cells via pattern recognition receptors. Toll-like receptor (TLR)-2 expression in macrophages is upregulated when exposed to the Gal/GalNAc lectin of E. histolytica, triggering pro-inflammatory cytokine production via NF-κB activation [12]. Macrophages that lack TLR-2 and TLR-4 displayed impaired response to E. histolytica lipopeptidophosphoglycan (LPPG), suggesting that pattern recognition is essential to the immune response [13]. Additionally, E. histolytica DNA can activate macrophages through interacting with TLR-9 [14]. Amebicidal activity of macrophages is contributed to by the production of nitric oxide (NO) from L-arginine, which is mediated by macrophage nitric oxide synthase. Inducible nitric oxide synthase (iNOS)–deficient mice were more susceptible to amebic liver abscess and to E. histolytica–induced hepatocytic apoptosis, implicating a critical role for NO in the host defense against amebiasis [15].

The Macrophage Migration Inhibitory Factor Homolog of Entamoeba histolytica Binds to and Immunomodulates Host Macrophages

ABSTRACT The host inflammatory response contributes to the tissue damage that occurs during amebic colitis, with tumor necrosis factor alpha (TNF-α) being a key mediator of the gut inflammation observed. Mammalian macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that plays an important role in the exacerbation of a wide range of inflammatory diseases, including colitis. We identified a MIF gene homolog in the Entamoeba histolytica genome, raising the question of whether E. histolytica MIF (EhMIF) has proinflammatory activity similar to that of mammalian MIF. In this report, we describe the first functional characterization of EhMIF. Antibodies were prepared against recombinantly expressed EhMIF and used to demonstrate that EhMIF is expressed as a 12-kDa protein localized to the cytoplasm of trophozoites. In a manner similar to that of mammalian MIF, EhMIF interacted with the MIF receptor CD74 and bound to macrophages. EhMIF induced interleukin-6 (IL-6) production. In addition, EhMIF enhanced TNF-α secretion by amplifying TNF-α production by lipopolysaccharide (LPS)-stimulated macrophages and by inhibiting the glucocorticoid-mediated suppression of TNF-α secretion. EhMIF was expressed during human infection, as evidenced by the presence of anti-EhMIF antibodies in the sera of children living in an area where E. histolytica infection is endemic. Anti-EhMIF antibodies did not cross-react with human MIF. The ability of EhMIF to modulate host macrophage function may promote an exaggerated proinflammatory immune response and contribute to the tissue damage seen in amebic colitis.

Fulminant Amebic Colitis after Corticosteroid Therapy: A Systematic Review.

Background Amebic colitis, caused by intestinal infection with the parasite, Entamoeba histolytica, is a common cause of diarrhea worldwide. Fulminant amebic colitis is the most devastating complication of this infection, associated with both high mortality and morbidity. We conducted a review of the English literature to describe cases of fulminant amebic colitis associated with exposure to corticosteroid medications in order to identify the risk factors for poor outcome and determine difficulties in diagnosis and treatment. Methodology and Principal Findings Articles reporting severe and fulminant forms of amebic colitis between 1991 and 2016 were collected. 525 records were screened to identify 24 cases for qualitative analysis associated with corticosteroid use. Cases arose from areas of high endemicity or travel to such areas. Most cases (14 of 24, 58%) were given corticosteroids for initially misdiagnosed colitis, mainly inflammatory bowel, resulting in rapid progression of disease. Nearly half of all cases underwent surgical intervention, and 25% of cases died, despite all patients eventually receiving treatment with metronidazole. The odds of death did not differ significantly by prior misdiagnosis, co-morbidities, bowel perforation or need for surgery. Conclusions and Significance Infection with E. histolytica should be considered prior to the administration of corticosteroids, in particular for patients residing in endemic areas or those with appropriate travel history, especially prior to the diagnosis of inflammatory bowel disease. The development of preventative and treatment interventions are needed to improve outcomes of fulminant disease.

Entamoeba Species in South Africa: Correlations With the Host Microbiome, Parasite Burdens, and First Description of Entamoeba bangladeshi Outside of Asia

Background Diarrhea is frequent in communities without clean water, which include low-income South African populations in Giyani and Pretoria. In these populations, the amount of diarrhea caused by Entamoeba histolytica, inclusive of all ages, sexes, and human immunodeficiency virus status, is uncertain. Infection with E. histolytica can modulate the host microbiota, and a key species indicative of this is the Prevotella copri pathobiont. Methods A cross-sectional study of patients attending gastroenterology clinics was conducted to determine the frequency and burden of 4 Entamoeba species and P. copri. Results Entamoeba species were present in 27% of patients (129/484), with E. histolytica detected in 8.5% (41), E. dispar in 8% (38), E. bangladeshi in 4.75% (23), and E. moshkovskii in 0%. This is the first description of E. bangladeshi outside Bangladesh. In E. histolytica-positive samples, the levels of both the parasite and P. copri were lower in nondiarrheal samples, validating the results of a study in Bangladesh (P = .0034). By contrast, in E. histolytica-negative samples positive for either of the nonpathogenic species E. dispar or E. bangladeshi, neither P. copri nor Entamoeba levels were linked to gastrointestinal status. Conclusions Nonmorphologic identification of this parasite is essential. In South Africa, 3 morphologically identical Entamoeba were common, but only E. histolytica was linked to both disease and changes in the microbiota.

Microbiome-mediated neutrophil recruitment via CXCR2 and protection from amebic colitis

The disease severity of Entamoeba histolytica infection ranges from asymptomatic to life-threatening. Recent human and animal data implicate the gut microbiome as a modifier of E. histolytica virulence. Here we have explored the association of the microbiome with susceptibility to amebiasis in infants and in the mouse model of amebic colitis. Dysbiosis occurred symptomatic E. histolytica infection in children, as evidenced by a lower Shannon diversity index of the gut microbiota. To test if dysbiosis was a cause of susceptibility, wild type C57BL/6 mice (which are innately resistant to E. histiolytica infection) were treated with antibiotics prior to cecal challenge with E. histolytica. Compared with untreated mice, antibiotic pre-treated mice had more severe colitis and delayed clearance of E. histolytica. Gut IL-25 and mucus protein Muc2, both shown to provide innate immunity in the mouse model of amebic colitis, were lower in antibiotic pre-treated mice. Moreover, dysbiotic mice had fewer cecal neutrophils and myeloperoxidase activity. Paradoxically, the neutrophil chemoattractant chemokines CXCL1 and CXCL2, as well as IL-1β, were higher in the colon of mice with antibiotic-induced dysbiosis. Neutrophils from antibiotic pre-treated mice had diminished surface expression of the chemokine receptor CXCR2, potentially explaining their inability to migrate to the site of infection. Blockade of CXCR2 increased susceptibility of control non-antibiotic treated mice to amebiasis. In conclusion, dysbiosis increased the severity of amebic colitis due to decreased neutrophil recruitment to the gut, which was due in part to decreased surface expression on neutrophils of CXCR2.

Entamoeba histolytica–Encoded Homolog of Macrophage Migration Inhibitory Factor Contributes to Mucosal Inflammation during Amebic Colitis

Understanding the mechanisms by which Entamoeba histolytica drives gut inflammation is critical for the development of improved preventive and therapeutic strategies. E. histolytica encodes a homolog of the human cytokine macrophage migration inhibitory factor (MIF). Here, we investigated the role of E. histolytica MIF (EhMIF) during infection. We found that the concentration of fecal EhMIF correlated with the level of intestinal inflammation in persons with intestinal amebiasis. Mice treated with antibodies that specifically block EhMIF had reduced chemokine expression and neutrophil infiltration in the mucosa. In addition to antibody-mediated neutralization, we used a genetic approach to test the effect of EhMIF on mucosal inflammation. Mice infected with parasites overexpressing EhMIF had increased chemokine expression, neutrophil influx, and mucosal damage. Together, these results uncover a specific parasite protein that increases mucosal inflammation, expands our knowledge of host-parasite interaction during amebic colitis, and highlights a potential immunomodulatory target.

Hemolytic Uremic Syndrome following Infection with O111 Shiga Toxin-Producing Escherichia coli Revealed through Molecular Diagnostics

ABSTRACT We report a case of hemolytic uremic syndrome in a 69-year-old woman due to Shiga toxin-producing Escherichia coli, possibly serotype O111, to illustrate the potentially deleterious implications of a Campylobacter enzyme immunoassay (EIA) result and the increasing importance of molecular testing when conventional methods are limited.

Erythrocyte lysis and Xenopus laevis oocyte rupture by recombinant Plasmodium falciparum hemolysin III.

ABSTRACT Malaria kills more than 1 million people per year worldwide, with severe malaria anemia accounting for the majority of the deaths. Malaria anemia is multifactorial in etiology, including infected erythrocyte destruction and decrease in erythrocyte production, as well as destruction or clearance of noninfected erythrocytes. We identified a panspecies Plasmodium hemolysin type III related to bacterial hemolysins. The identification of a hemolysin III homologue in Plasmodium suggests a potential role in host erythrocyte lysis. Here, we report the first characterization of Plasmodium falciparum hemolysin III, showing that the soluble recombinant P. falciparum hemolysin III is a pore-forming protein capable of lysing human erythrocytes in a dose-, time-, and temperature-dependent fashion. The recombinant P. falciparum hemolysin III-induced hemolysis was partially inhibited by glibenclamide, a known channel antagonist. Studies with polyethylene glycol molecules of different molecular weights indicated a pore size of approximately 3.2 nm. Heterologous expression of recombinant P. falciparum hemolysin III in Xenopus oocytes demonstrated early hypotonic lysis similar to that of the pore-forming aquaporin control. Live fluorescence microscopy localized transfected recombinant green fluorescent protein (GFP)-tagged P. falciparum hemolysin III to the essential digestive vacuole of the P. falciparum parasite. These transfected trophozoites also possessed a swollen digestive vacuole phenotype. Native Plasmodium hemolysin III in the digestive vacuole may contribute to lysis of the parasitophorous vacuole membrane derived from the host erythrocyte. After merozoite egress from infected erythrocytes, remnant P. falciparum hemolysin III released from digestive vacuoles could potentially contribute to lysis of uninfected erythrocytes to contribute to severe life-threatening anemia.

The Impact of Systemic Inflammation on Neurodevelopment

Inflammatory mediators affect the brain during development. Neurodevelopmental disorders such as autism spectrum disorders, cognitive impairment, cerebral palsy, epilepsy, and schizophrenia have been linked to early life inflammation. Recent advances have shown the effects of systemic inflammation on childrens neurodevelopment. We discuss the potential mechanisms by which inflammatory molecules can exert their effects on the developing brain and consider the roles of MHC class I molecules, the HPA axis, glial cells, and monoamine metabolism. Methods to prevent the effects of cytokine imbalance may lead to the development of new therapeutics for neuropsychiatric disorders. Future research should focus on identifying at-risk individuals and early effective interventions to prevent long-term neurodevelopmental disabilities.