Entamoeba histolytica cysteine proteases cleave the MUC2 mucin in its C-terminal domain and dissolve the protective colonic mucus gel

In order for the protozoan parasite Entamoeba histolytica (E.h.) to cause invasive intestinal and extraintestinal infection, which leads to significant morbidity and mortality, it must disrupt the protective mucus layer by a previously unknown mechanism. We hypothesized that cysteine proteases secreted from the amoeba disrupt the mucin polymeric network, thereby overcoming the protective mucus barrier. The MUC2 mucin is the major structural component of the colonic mucus gel. Heavily O-glycosylated and protease-resistant mucin domains characterize gel-forming mucins. Their N- and C-terminal cysteine-rich domains are involved in mucin polymerization, and these domains are likely to be targeted by proteases because they are less glycosylated, thereby exposing their peptide chains. By treating recombinant cysteine-rich domains of MUC2 with proteases from E.h. trophozoites, we showed that the C-terminal domain was specifically targeted at two sites by cysteine proteases, whereas the N-terminal domain was resistant to proteolysis. The major cleavage site is predicted to depolymerize the MUC2 polymers, thereby disrupting the protective mucus gel. The ability of the cysteine proteases to dissolve mucus gels was confirmed by treating mucins from a MUC2-producing cell line with amoeba proteases. These findings suggest a major role for E.h. cysteine proteases in overcoming the protective mucus barrier in the pathogenesis of invasive amoebiasis. In this report, we identify a specific cleavage mechanism used by an enteric pathogen to disrupt the polymeric nature of the mucin gel.     

Hereditary hemochromatosis

Hereditary hemochromatosis is a relatively common genetic disorder characterized by excess dietary iron absorption and deposition in tissues with resulting end-organ damage. Early diagnosis and initiation of therapeutic phlebotomy can provide a normal life expectancy for affected individuals.     

Entamoeba histolytica Infection and Secreted Proteins Proteolytically Damage Enteric Neurons

The enteric protozoan parasite Entamoeba histolytica causes amebic colitis through disruption of the mucus layer, followed by binding to and destruction of epithelial cells. However, it is not known whether ameba infections or ameba components can directly affect the enteric nervous system. Analysis of mucosal innervations in the mouse model of cecal amebiasis showed that axon density was diminished to less than 25% of control. To determine whether amebas directly contributed to axon loss, we tested the effect of either E. histolytica secreted products (Eh-SEC) or soluble components (Eh-SOL) to an established coculture model of myenteric neurons, glia, and smooth muscle cells. Neuronal survival and axonal degeneration were measured after 48 h of exposure to graded doses of Eh-SEC or Eh-SOL (10 to 80 μg/ml). The addition of 80 μg of either component/ml decreased the neuron number by 30%, whereas the axon number was decreased by 50%. Cytotoxicity was specific to the neuronal population, since the glial and smooth muscle cell number remained similar to that of the control, and was completely abrogated by prior heat denaturation. Neuronal damage was partially prevented by the cysteine protease inhibitor E-64, showing that a heat-labile protease was involved. E. histolytica lysates derived from amebas deficient in the major secreted protease EhCP5 caused a neurotoxicity similar to that of wild-type amebas. We conclude that E. histolytica infection and ameba protease activity can cause selective damage to enteric neurons.Entamoeba histolytica is a protozoan enteric parasite of humans that colonizes the colon, where it typically causes asymptomatic luminal infections. However, in ca. 10% of individuals, the parasite invades the mucosa to cause amoebic colitis, characterized by ulcerative lesions, diarrhea, and fever and, in severe cases can disseminate to soft organs (39). Although normally seldom fatal, the consequences of ameba infection become significant in the immunosuppressed. The mechanism of infection in the intestine is complex and involves dissolution of the mucus layer by motile trophozoites, followed by adhesion and lysis of epithelial cells and invading leukocytes (8, 9).Cysteine proteases are important in the differentiation and pathogenicity of E. histolytica, and its genome contains about 50 genes coding for cysteine peptidases (36). A number of in vivo and in vitro studies have implicated cysteine protease activity as a major mechanism of cell death of infected cells, as well as degradation of the extracellular matrix and activation of the complement system (31). Although cell-cell contact is thought to be required for intestinal invasion by E. histolytica trophozoites, amebic proteins have been shown to cause cellular responses in vitro. For example, E. histolytica trophozoite-secreted products caused mucin degradation by proteolytic degradation of cysteine domains (28). In addition, incubation of secreted products and soluble proteins with cultured intestinal epithelial cells resulted in the upregulation of interleukin-8 mRNA to a similar extent as live trophozoites (11, 41). This suggests that both secreted products and direct contact serve important roles in the course of infection.The consequences to the enteric nervous system (ENS) of E. histolytica infection are unknown but may constitute an important part of its pathogenicity. The epithelial layer of the intestine is innervated by axons extending from the submucosal ganglia of the ENS. This innervation is structurally and functionally poised to respond to factors affecting the integrity of the epithelial barrier, such as amoebic invasion and the release of cysteine proteases. In addition, intestinal inflammation can lead to permanent damage to the enteric nervous system in human disease, as well as in animal models. Neuronal hypertrophy and myenteric and submucosal plexitis are among the featured characteristics observed in patients with Crohn''s disease (12), and models of colitis in the rat and other rodents show neuronal death and axonal degeneration in both the myenteric and the submucosal plexuses (24, 34). Since inflammation of the colon due to amebic invasion can resemble that seen in inflammatory bowel disease (30), we hypothesized that the enteric nervous system will also be damaged during amebic colitis.To study this, we analyzed the effect of E. histolytica infection on axon integrity in an established model of invasive murine cecal amebiasis (18). The infected intestine showed a substantial decrease in axon number compared to the control, which was inversely correlated with the extent of tissue damage. We also used an in vitro model of intestinal neurons, smooth muscle, and glia (23) and examined the effects of either amebic secreted products (Eh-SEC) or soluble components (Eh-SOL) on neuronal survival and axonal structure. We found that a population of enteric neurons was targeted by Entamoeba-derived cysteine proteases, leading to selective damage in vitro. This shows that the innervation of the mucosa by the ENS is a potential target of E. histolytica intestinal invasion.     

Nonhygienic behavior, knowledge, and attitudes among interactive splash park visitors

Nonhygienic behavior likely contributed to three recreational waterborne illness (RWI) outbreaks at Idaho splash parks. The study described in this article examined the influence of signage and hygiene attendant presence on rates of nonhygienic behavior among children at splash parks and knowledge and attitudes of their adult supervisors. Investigators observed children for nonhygienic behaviors at four Idaho splash parks, two with signage and attendants. Supervisors were surveyed (N = 551) using an eight-item survey. Individually observed children (N = 145) were often seen exposing their buttocks to splash feature water and placing an open mouth to water. The rate of nonhygienic behaviors was not lower at parks with signage or staff. Supervisors reported bathing children before splash park entry infrequently. Signage and hygiene attendants do not adequately limit nonhygienic behaviors at splash parks, and supervisors have insufficient understanding of RWI. These findings have implica.tions for developing splash park regulations and RWI prevention efforts.     

Ethical principles and legal requirements for pediatric research in the EU: an analysis of the European normative and legal framework surrounding pediatric clinical trials

The involvement of minors in clinical research is inevitable to catch up with the lack of drugs labeled for pediatric use. To encourage the responsible conduct of pediatric clinical trials in the EU, an extensive legal framework has been developed over the past decade in which the practical, ethical, legal, social, and commercial issues in pediatric research are addressed. In this article, the European legal framework surrounding pediatric clinical trials is analyzed from the perspective of the major ethical concerns in pediatric research. The four principles of biomedical ethics will be used as a conceptual framework (1) to map the ethical issues addressed in the European legal framework, (2) to study how these issues are commonly handled in competent adults, (3) to detect workability problems of these paradigmatic approaches in the specific setting of pediatric research, and (4) to illustrate the strong urge to differentiate, specify, or adjust these paradigmatic approaches to guarantee their successful operation in pediatric research. In addition, a concise comparative analysis of the European regulation will be made. To conclude our analysis, we integrate our findings in the existing ethical discussions on issues specific to pediatric clinical research.

Pharmacodynamics of cidofovir for vaccinia virus infection in an in vitro hollow-fiber infection model system

Variola major virus remains a potent weapon of bioterror. There is currently an investigational-new-drug application for cidofovir for the therapy of variola major virus infections. Stittelaar and colleagues compared the levels of effectiveness of postexposure smallpox vaccination (Elstree-RIVM) and antiviral treatment with cidofovir or an acyclic nucleoside phosphonate analogue {6-[2-(phosphonomethoxy)alkoxy]-2,4-diaminopyrimidine (HPMPO-DAPy)} after lethal intratracheal infection of cynomolgus monkeys with monkeypox virus, a variola virus surrogate. Their results demonstrated that either compound was more effective than vaccination with the Ellstree vaccine (K. J. Stittelaar et al., Nature 439:745-748, 2006). An unanswered question is how to translate this information into therapy for poxvirus infections in people. In a proof-of-principle study, we used a novel in vitro hollow-fiber infection model system to determine the pharmacodynamics of vaccinia virus infection of HeLa-S3 cells treated with cidofovir. Our results demonstrate that the currently licensed dose of cidofovir of 5 mg/kg of body weight weekly with probenecid (which ameliorates nephrotoxicity) is unlikely to provide protection for patients intentionally exposed to Variola major virus. We further demonstrate that the antiviral effect is independent of the schedule of drug administration. Exposures (area under the concentration-time curve) to cidofovir that will have a robust protective effect will require doses that are 5 to 10 times that currently administered to humans. Such doses may cause nephrotoxicity, and therefore, approaches that include probenecid administration as well as schedules of administration that will help ameliorate the uptake of cidofovir into renal tubular epithelial cells need to be considered when addressing such treatment for people.     

Theranostics by testing CFTR modulators in patient-derived materials: The current status and a proposal for subjects with rare CFTR mutations

The last decade has witnessed developments in the CF drug pipeline which are both exciting and unprecedented, bringing with them previously unconsidered challenges. The Task Force group came together to consider these challenges and possible strategies to address them. Over the last 18 months, we have discussed internally and gathered views from a broad range of individuals representing patient organizations, clinical and research teams, the pharmaceutical industry and regulatory agencies. In this and the accompanying article, we discuss two main areas of focus: i) optimising trial design and delivery for speed/efficiency; ii) drug development for patients with rare CFTR mutations. We propose some strategies to tackle the challenges ahead and highlight areas where further thought is needed. We see this as the start of a process rather than the end and hope herewith to engage the wider community in seeking solutions to improved treatments for all patients with CF.     

Speeding up access to new drugs for CF: Considerations for clinical trial design and delivery

The last decade has witnessed developments in the CF drug pipeline which are both exciting and unprecedented, bringing with them previously unconsidered challenges. The Task Force group was brought together to consider these challenges and possible strategies to address them. Over the last 18 months, we have discussed internally and gathered views from a broad range of individuals representing patient organisations, clinical and research teams, the pharmaceutical industry and regulatory agencies. In this and the accompanying article, we discuss two main areas of focus: i) optimising trial design and delivery for speed and efficiency; ii) drug development for patients with rare CFTR mutations. We propose some strategies to tackle the challenges ahead and highlight areas where further thought is needed. We see this as the start of a process rather than the end and hope herewith to engage the wider community in seeking solutions to improved treatments for all patients with CF.