Human Intestinal Infections Research Articles

Bacteriophage-fused peptides for serodiagnosis of human strongyloidiasis.

BackgroundStrongyloidiasis, a human intestinal infection caused by the nematode Strongyloides stercoralis, is frequently underdiagnosed and although its high prevalence is still a neglected parasitic disease because conventional diagnostic tests based on parasitological examination (presence of Strongyloides larvae in stool) are not sufficiently sensitive due to the low parasitic load and to the irregular larval output. There is an urgent need to improve diagnostic assays, especially for immunocompromised patients with high parasitic load as consequence of self-infection cycle, which can disseminate throughout the body, resulting in a potentially fatal hyperinfection syndrome often accompanied by sepsis or meningitis.Methods/Principal FindingsWe have performed Phage Display technology to select peptides that mimic S. stercoralis antigens, capable of detecting a humoral response in patients with strongyloidiasis. The peptides reactivity was investigated by Phage-ELISA through different panels of serum samples. We have successfully selected five peptides with significant immunoreactivity to circulating IgG from patients' sera with strongyloidiasis. The phage displayed peptides C9 and C10 presented the highest diagnostic potential (AUC>0.87) with excellent sensitivity (>85%) and good specificity (>77.5%), suggesting that some S. stercoralis antigens trigger systemic immune response.Conclusions/SignificanceThese novel antigens are interesting serum biomarkers for routine strongyloidiasis screenings due to the easy production and simple assay using Phage-ELISA. Such markers may also present a promising application for therapeutic monitoring.

Effect of sampling and diagnostic effort on the assessment of schistosomiasis and soil-transmitted helminthiasis and drug efficacy: a meta-analysis of six drug efficacy trials and one epidemiological survey.

It is generally recommended to perform multiple stool examinations in order to improve the diagnostic accuracy when assessing the impact of mass drug administration programmes to control human intestinal worm infections and determining efficacy of the drugs administered. However, the collection and diagnostic work-up of multiple stool samples increases costs and workload. It has been hypothesized that these increased efforts provide more accurate results when infection and drug efficacy are summarized by prevalence (proportion of subjects infected) and cure rate (CR, proportion of infected subjects that become egg-negative after drug administration), respectively, but not when these indicators are expressed in terms of infection intensity and egg reduction rate (ERR). We performed a meta-analysis of six drug efficacy trials and one epidemiological survey. We compared prevalence and intensity of infection, CR and ERR based on collection of one or two stool samples that were processed with single or duplicate Kato-Katz thick smears. We found that the accuracy of prevalence estimates and CR was lowest with the minimal sampling effort, but that this was not the case for estimating infection intensity and ERR. Hence, a single Kato-Katz thick smear is sufficient for reporting infection intensity and ERR following drug treatment.

Towards an understanding of the role of Clostridium perfringens toxins in human and animal disease.

Clostridium perfringens uses its arsenal of >16 toxins to cause histotoxic and intestinal infections in humans and animals. It has been unclear why this bacterium produces so many different toxins, especially since many target the plasma membrane of host cells. However, it is now established that C. perfringens uses chromosomally encoded alpha toxin (a phospholipase C) and perfringolysin O (a pore-forming toxin) during histotoxic infections. In contrast, this bacterium causes intestinal disease by employing toxins encoded by mobile genetic elements, including C. perfringens enterotoxin, necrotic enteritis toxin B-like, epsilon toxin and beta toxin. Like perfringolysin O, the toxins with established roles in intestinal disease form membrane pores. However, the intestinal disease-associated toxins vary in their target specificity, when they are produced (sporulation vs vegetative growth), and in their sensitivity to intestinal proteases. Producing many toxins with diverse characteristics likely imparts virulence flexibility to C. perfringens so it can cause an array of diseases.

Campylobacter fetus infection presenting with bacteremia and cellulitis in a 72-year-old man with an implanted pacemaker: a case report

IntroductionCampylobacter is an important causative agent of intestinal infections in humans. Bacteremia is detected in less than 1% of patients, mainly in immunocompromised patients and in extreme age groups. Cellulitis is a relatively common manifestation of Campylobacter infection, but concomitant bacteremia is a rare event. Infections of the pacemaker area are caused primarily by staphylococci, followed by fungi, streptococci and Gram-negative rods. To the best of our knowledge, this is the first case report of pacemaker pocket infection and bacteremia caused by Campylobacter fetus.Case presentationA 72-year-old Croatian Caucasian man with myelodysplasia, impaired fasting glucose levels and a recently implanted permanent pacemaker was admitted to hospital after six days of fever, development of red swelling of the pacemaker pocket area and worsening of his general condition. No antibiotic therapy was introduced in the outpatient setting. He denied any recent gastrointestinal disturbances. With the exception of an elevated leukocyte count, erythrocyte sedimentation rate, and C-reactive protein and blood glucose levels, other laboratory findings were normal. Treatment with vancomycin plus netilmicin was introduced, and a surgical incision with drainage of the pacemaker pocket was performed. The entire pacemaker system was removed and a new one re-implanted after 14 days of antibiotic therapy. Transesophageal echocardiography showed no pathological findings. Three subsequent blood cultures obtained on admission as well as swab culture of the incised pacemaker area revealed Campylobacter fetus; stool and pacemaker lead cultures were negative. According to the microbiological results, antibiotic therapy was changed to ciprofloxacin plus netilmicin. A clinical examination and the results of a laboratory analysis performed after two weeks of therapy were within normal limits.ConclusionMyelodysplasia, impaired fasting glucose levels and older age could be contributing factors for the development of bacteremic Campylobacter fetus cellulitis. Emergent surgical and antibiotic treatment are mandatory and provide the optimal outcome for such types of pacemaker pocket infection.

Evidence of Microbial Translocation Associated with Perturbations in T Cell and Antigen-Presenting Cell Homeostasis in Hookworm Infections

BackgroundMicrobial translocation (MT) is the process by which microbes or microbial products translocate from the intestine to the systemic circulation. MT is a common cause of systemic immune activation in HIV infection and is associated with reduced frequencies of CD4+ T cells; no data exist, however, on the role of MT in intestinal helminth infections.MethodsWe measured the plasma levels of MT markers, acute-phase proteins, and pro- and anti - inflammatory cytokines in individuals with or without hookworm infections. We also estimated the absolute counts of CD4+ and CD8+ T cells as well as the frequencies of memory T cell and dendritic cell subsets. Finally, we also measured the levels of all of these parameters in a subset of individuals following treatment of hookworm infection.ResultsOur data suggest that hookworm infection is characterized by increased levels of markers associated with MT but not acute-phase proteins nor pro-inflammatory cytokines. Hookworm infections were also associated with increased levels of the anti – inflammatory cytokine – IL-10, which was positively correlated with levels of lipopolysaccharide (LPS). In addition, MT was associated with decreased numbers of CD8+ T cells and diminished frequencies of particular dendritic cell subsets. Antihelmintic treatment of hookworm infection resulted in reversal of some of the hematologic and microbiologic alterations.ConclusionsOur data provide compelling evidence for MT in a human intestinal helminth infection and its association with perturbations in the T cell and antigen-presenting cell compartments of the immune system. Our data also reveal that at least one dominant counter-regulatory mechanism i.e. increased IL-10 production might potentially protect against systemic immune activation in hookworm infections.

Human Milk Mucin 1 and Mucin 4 Inhibit Salmonella enterica Serovar Typhimurium Invasion of Human Intestinal Epithelial Cells In Vitro1–3

Many human milk glycans inhibit pathogen binding to host receptors and their consumption by infants is associated with reduced risk of disease. Salmonella infection is more frequent among infants than among the general population, but the incidence is lower in breast-fed babies, suggesting that human milk could contain components that inhibit Salmonella. This study aimed to test whether human milk per se inhibits Salmonella invasion of human intestinal epithelial cells in vitro and, if so, to identify the milk components responsible for inhibition. Salmonella enterica serovar Typhimurium SL1344 (SL1344) invasion of FHs 74 Int and Caco-2 cells were the models of human intestinal epithelium infection. Internalization of fluorescein-5-isothiocyanate–labeled SL1344 into intestinal cells was measured by flow cytometry to quantify infection. Human milk and its fractions inhibited infection; the inhibitory activity localized to the high molecular weight glycans. Mucin 1 and mucin 4 were isolated to homogeneity. At 150 μg/L, a typical concentration in milk, human milk mucin 1 and mucin 4 inhibited SL1344 invasion of both target cell types. These mucins inhibited SL1344 invasion of epithelial cells in a dose-dependent manner. Thus, mucins may prove useful as a basis for developing novel oral prophylactic and therapeutic agents that inhibit infant diseases caused by Salmonella and related pathogens.

Campylobacter jejuni-Mediated Induction of CC and CXC Chemokines and Chemokine Receptors in Human Dendritic Cells

Campylobacter jejuni is a leading worldwide bacterial cause of human diarrheal disease. Although the specific molecular mechanisms of C. jejuni pathogenesis have not been characterized in detail, host inflammatory responses are thought to be major contributing factors to the resulting typical acute colitis. The intestinal mucosal chemokine response is particularly important in the initial stages of bacterium-induced gut inflammation. Chemokines attract blood phagocytes and lymphocytes to the site of infection and regulate immune cell maturation and the development of localized lymphoid tissues. The production of chemokines by dendritic cells (DCs) following Campylobacter infection has not yet been analyzed. In the current study, we infected human monocyte-derived DCs with C. jejuni to examine the production of key proinflammatory chemokines and chemokine receptors. The chemokines, including CC families (macrophage inflammatory protein 1α [MIP-1α], MIP-1β, RANTES) and CXC families (growth-related oncogene α [GRO-α], IP-10, and monokine induced by gamma interferon [MIG]), were upregulated in Campylobacter-infected DCs. Chemokine receptors CCR6 and CCR7, with roles in DC trafficking, were also induced in Campylobacter-infected DCs. Further, Campylobacter infection stimulated the phosphorylation of P38, P44/42, and stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK) mitogen-activated protein kinases (MAPKs) in DCs. NF-κB activation was specifically involved in chemokine induction in DCs infected with C. jejuni. Additionally, STAT3 was significantly increased in Campylobacter-infected DCs compared to that in uninfected DCs. These results suggest that DCs play a significant role in the initiation and modulation of the inflammatory response by enlisting monocytes, neutrophils, and T lymphocytes during human intestinal infection with Campylobacter.

Additive Function of Vibrio vulnificus MARTXVv and VvhA Cytolysins Promotes Rapid Growth and Epithelial Tissue Necrosis During Intestinal Infection

Vibrio vulnificus is a pathogen that causes both severe necrotizing wound infections and life-threatening food-borne infections. Food-borne infection is particularly lethal as the infection can progress rapidly to primary septicemia resulting in death from septic shock and multiorgan failure. In this study, we use both bioluminescence whole animal imaging and V. vulnificus bacterial colonization of orally infected mice to demonstrate that the secreted multifunctional-autoprocessing RTX toxin (MARTXVv) and the cytolysin/hemolysin VvhA of clinical isolate CMCP6 have an important function in the gut to promote early in vivo growth and dissemination of this pathogen from the small intestine to other organs. Using histopathology, we find that both cytotoxins can cause villi disruption, epithelial necrosis, and inflammation in the mouse small intestine. A double mutant deleted of genes for both cytotoxins was essentially avirulent, did not cause intestinal epithelial tissue damage, and was cleared from infected mice by 36 hours by an effective immune response. Therefore, MARTXVv and VvhA seem to play an additive role for pathogenesis of CMCP6 causing intestinal tissue damage and inflammation that then promotes dissemination of the infecting bacteria to the bloodstream and other organs. In the absence of these two secreted factors, we propose that this bacterium is unable to cause intestinal infection in humans.

In Vitro and In Vivo Efficacy of Monepantel (AAD 1566) against Laboratory Models of Human Intestinal Nematode Infections

BackgroundFew effective drugs are available for soil-transmitted helminthiases and drug resistance is of concern. In the present work, we tested the efficacy of the veterinary drug monepantel, a potential drug development candidate compared to standard drugs in vitro and in parasite-rodent models of relevance to human soil-transmitted helminthiases.MethodologyA motility assay was used to assess the efficacy of monepantel, albendazole, levamisole, and pyrantel pamoate in vitro on third-stage larvae (L3) and adult worms of Ancylostoma ceylanicum, Necator americanus and Trichuris muris. Ancylostoma ceylanicum- or N. americanus-infected hamsters, T. muris- or Ascaris suum-infected mice, and Strongyloides ratti-infected rats were treated with single oral doses of monepantel or with one of the reference drugs.Principal FindingsMonepantel showed excellent activity on A. ceylanicum adults (IC50 = 1.7 µg/ml), a moderate effect on T. muris L3 (IC50 = 78.7 µg/ml), whereas no effect was observed on A. ceylanicum L3, T. muris adults, and both stages of N. americanus. Of the standard drugs, levamisole showed the highest potency in vitro (IC50 = 1.6 and 33.1 µg/ml on A. ceylanicum and T. muris L3, respectively). Complete elimination of worms was observed with monepantel (10 mg/kg) and albendazole (2.5 mg/kg) in A. ceylanicum-infected hamsters. In the N. americanus hamster model single 10 mg/kg oral doses of monepantel and albendazole resulted in worm burden reductions of 58.3% and 100%, respectively. Trichuris muris, S. ratti and A. suum were not affected by treatment with monepantel in vivo (following doses of 600 mg/kg, 32 mg/kg and 600 mg/kg, respectively). In contrast, worm burden reductions of 95.9% and 76.6% were observed following treatment of T. muris- and A. suum infected mice with levamisole (200 mg/kg) and albendazole (600 mg/kg), respectively.Conclusions/SignificanceMonepantel reveals low or no activities against N. americanus, T. muris, S. ratti and A. suum in vivo, hence does not qualify as drug development candidate for human soil-transmitted helminthiases.

Change in Conformation with Reduction of α-Helix Content Causes Loss of Neutrophil Binding Activity in Fully Cytotoxic Shiga Toxin 1

Shiga toxins (Stx) play an important role in the pathogenesis of hemolytic uremic syndrome, a life-threatening renal sequela of human intestinal infection caused by specific Escherichia coli strains. Stx target a restricted subset of human endothelial cells that possess the globotriaosylceramide receptor, like that in renal glomeruli. The toxins, composed of five B chains and a single enzymatic A chain, by removing adenines from ribosomes and DNA, trigger apoptosis and the production of pro-inflammatory cytokines in target cells. Because bacteria are confined to the gut, the toxins move to the kidney through the circulation. Polymorphonuclear leukocytes (PMN) have been indicated as the carriers that "piggyback" shuttle toxins to the kidney. However, there is no consensus on this topic, because not all laboratories have been able to reproduce the Stx/PMN interaction. Here, we demonstrate that conformational changes of Shiga toxin 1, with reduction of α-helix content and exposition to solvent of hydrophobic tryptophan residues, cause a loss of PMN binding activity. The partially unfolded toxin was found to express both enzymatic and globotriaosylceramide binding activities being fully active in intoxicating human endothelial cells; this suggests the presence of a distinct PMN-binding domain. By reviewing functional and structural data, we suggest that A chain moieties close to Trp-203 are recognized by PMN. Our findings could help explain the conflicting results regarding Stx/PMN interactions, especially as the groups reporting positive results obtained Stx by single-step affinity chromatography, which could have preserved the correct folding of Stx with respect to more complicated multi-step purification methods.

Immunization with the recombinant antigen Ss-IR induces protective immunity to infection with Strongyloides stercoralis in mice

Human intestinal infections with the nematode Strongyloides stercoralis remain a significant problem worldwide and a vaccine would be a useful addition to the tools available to prevent and control this infection. The goal of this study was to test single antigens for their efficacy in a vaccine against S. stercoralis larvae in mice. Alum was used as the adjuvant in these studies and antigens selected for analysis were either recognized by protective human IgG (Ss-TMY-1, Ss-EAT-6, and Ss-LEC-5) or were known to be highly immunogenic in humans (Ss-NIE-1 and Ss-IR). Only mice immunized with the Ss-IR antigen demonstrated a significant decrease of approximately 80% in the survival of larval parasites in the challenge infection. Antibodies, recovered from mice with protective immunity to S. stercoralis after immunization with Ss-IR, were used to locate the antigen in the larvae. Confocal microscopy revealed that IgG from mice immunized with Ss-IR bound to the surface of the parasites and observations by electron microscopy indicated that IgG bound to granules in the glandular esophagus. Serum collected from mice immunized with Ss-IR passively transferred immunity to naïve mice. These studies demonstrate that Ss-IR, in combination with alum, induces high levels of protective immunity through an antibody dependent mechanism and may therefore be suitable for further development as a vaccine against human strongyloidiasis.

Comparison of the Flotac-400 Dual Technique and the Formalin-Ether Concentration Technique for Diagnosis of Human Intestinal Protozoon Infection

There is a need for accurate diagnosis of intestinal parasite infections in humans, but currently available copromicroscopic techniques have shortcomings, such as low sensitivity. We compared the diagnostic accuracy of a further modified version of the recently developed Flotac technique with that of the widely used formalin-ether concentration technique (FECT) for the detection of intestinal protozoa in human stool samples. Formaldehyde-preserved stool samples from 108 individuals in Côte d'Ivoire were subjected to the Flotac-400 dual technique, using two different flotation solutions (FS), and to the FECT. Stool samples were examined according to computer-generated random lists by an experienced laboratory technician blinded for the results of each method. Both methods detected the same eight intestinal protozoon species. While the Flotac-400 dual technique (results from both FS combined) found higher prevalences of Entamoeba coli (77.8% versus 71.3%, P < 0.001), Blastocystis hominis (20.4% versus 13.0%, P = 0.458), and Giardia intestinalis (8.3% versus 6.5%, P < 0.001), the FECT detected higher prevalences of Entamoeba histolytica/Entamoeba dispar (27.8% versus 20.4%, P = 0.019) and four species of nonpathogenic intestinal protozoa. The diagnostic agreement between the two methods differed considerably depending on the intestinal protozoon investigated (Cohen's kappa measures; range, 0.01 to 0.46). Our study confirmed that the Flotac-400 dual technique can be utilized for the diagnosis of intestinal protozoon infections in humans. Since Flotac is a sensitive technique for the detection of soil-transmitted helminths and Schistosoma mansoni, it might become a viable copromicroscopic technique for the concurrent diagnosis of helminths and intestinal protozoon infections.

Human milk mucins inhibit salmonella typhimurium invasion of human epithelial cells

Salmonella infection is more frequent among infants than the general population, but the incidence is lower in breastfed babies. This study was to test whether human milk per se inhibited salmonella invasion of human intestinal epithelial cells in vitro, and to identify the milk components likely to be responsible for any inhibition. Salmonella typhimurium SL1344 invasion of FHs 74 Int and CaCo‐2 cells were the models of human intestinal epithelium infection. Internalization of FITC‐labeled salmonella into intestinal cells was measured by FACS analysis to quantify infection. Pooled human milk and its fractions were tested for their ability to inhibit infection, including two human milk mucins, mucin 1 and mucin 4. At 150 ng/mL, approximating the amount in milk, human milk mucin 1 and mucin 4 inhibited salmonella invasion of both cell types. They also bound Salmonella typhimurium SL1344 in a dose‐dependent manner. Thus, mucins may prove useful as a basis for the development of novel oral prophylactic and therapeutic agents that inhibit infant diseases caused by salmonella, and perhaps other pathogens. Supported by NIH HD013021 and AI075563.

Drosophila melanogaster as a model for human intestinal infection and pathology

Recent findings concerning Drosophila melanogaster intestinal pathology suggest that this model is well suited for the study of intestinal stem cell physiology during aging, stress and infection. Despite the physiological divergence between vertebrates and insects, the modeling of human intestinal diseases is possible in Drosophila because of the high degree of conservation between Drosophila and mammals with respect to the signaling pathways that control intestinal development, regeneration and disease. Furthermore, the genetic amenability of Drosophila makes it an advantageous model species. The well-studied intestinal stem cell lineage, as well as the tools available for its manipulation in vivo, provide a promising framework that can be used to elucidate many aspects of human intestinal pathology. In this Perspective, we discuss recent advances in the study of Drosophila intestinal infection and pathology, and briefly review the parallels and differences between human and Drosophila intestinal regeneration and disease.

Rapid appraisal of human intestinal helminth infections among schoolchildren in Osh oblast, Kyrgyzstan

A population-representative lot quality assurance sampling (LQAS) survey was conducted in 2009 to determine the prevalence of intestinal helminth infections among schoolchildren across Osh oblast, Kyrgyzstan. The diagnostic approach consisted of duplicate Kato-Katz thick smears from a single stool sample and an adhesive tape test. A questionnaire was administered to identify risk factors for infections. A total of 1262 schoolchildren aged 6–15 years were recruited; 41% of them harboured at least one of the eight identified helminth species. The two most prevalent helminths were Ascaris lumbricoides (23.1%) and Enterobius vermicularis (19.3%). Lower prevalences were found for Hymenolepis nana (4.4%), Fasciola hepatica (1.9%) and Dicrocoelium dendriticum (1.8%). Washing raw vegetables was a protective factor with regard to A. lumbricoides infection (odds ratio (OR) = 0.69, p = 0.022); tap water was borderline protective (OR = 0.56, p = 0.057). Children of the richest families were at a lower risk of E. vermicularis infection than the poorest ones (OR = 0.41, p = 0.011). Sharing the bed with more than one person was a risk factor for E. vermicularis infection (OR = 2.0, p = 0.002). The results call for targeted interventions against intestinal helminths in Osh oblast. In a first stage, annual deworming of schoolchildren and other high-risk groups with albendazole or mebendazole should be implemented, and reliable diagnosis and additional anthelminthic drugs should be made available. Subsequently, transmission control including locally-adapted health education, improved water supply and adequate sanitation should become the central features.

Intestinal Infections in Humans in the Rocky Mountain Region, United States

To evaluate the seasonal prevalence of human intestinal parasites in the western states of Colorado, Utah, New Mexico, and Montana, fecal samples were examined as part of routine diagnostic testing from patients experiencing gastrointestinal discomfort in August (summer) 2006, January (winter), and April (spring) 2007. Parasite identification in positive samples was confirmed using light microscopy after wet mount and trichrome staining techniques. Seventy-eight of the 1,083 patients surveyed (7.2%) in August tested positive for at least 1 species of intestinal parasite. Forty-eight of 726 (6.6%) patients and 51 of 795 (6.4%) patients tested positive for at least 1 species in January and April, respectively. Blastocystis sp. was the most prevalent, followed by Giardia lamblia. Approximately 25% of the parasite occurrences were multiple infections involving fecal-oral transmitted species. Co-infections with Entamoeba spp. and Blastocystis sp. were common, suggesting a possible fecal-oral transmission for the latter parasite. Entamoeba spp. were more likely to co-occur than independently. Other species detected included Endolimax nana, Diphyllobothrium latum, Hymenolepis nana, Dientamoeba fragilis, and Iodamoeba butschlii.

The incidence, intensity and host morbidity of human parasitic protozoan infections in gastrointestinal disorder outpatients in Buea Sub Division, Cameroon

Numerous protozoans inhibit the gastrointestinal tract of humans with the majority being either non-pathogenic commensals or of a type that may result in mild disease. However, some of these organisms can cause severe diseases under certain circumstances while others may become highly virulent and invasive causing potentially lethal systemic disease. This study investigated the prevalence, intensity and host morbidity of human intestinal protozoan infections in individuals living in the Buea Sub-Division, Cameroon. Random sampling was used to collect stool samples from 356 patients in a cross-sectional study. All samples were examined by formol-ether concentration and direct smear techniques. Data collected was analyzed and differences in proportions were determined using the Chi square (chi(2)) test, Fisher's exact test, or analysis of variance where appropriate. It was found that 28.1% (100/356) of the sampled population were infected with protozoans. Females showed a higher infection rate (29.7%; 56/182) than males (26.4%; 46/174) and there was a significantly (P < 0.001) higher prevalence in rural areas (38.7%; 55/142) than in urban areas (21.0%; 45/214). The 6 to 12 years age group had a significantly (P < 0.05) higher infection rate (42.9%; 30/70). The total prevalence of intestinal protozoans was as follows: E. histolytica (24.4%), E. coli (11.2%) and G. lamblia (0.6%). The most prevalent morbidity effects associated with intestinal protozoan infections were abdominal pains, dysentery and body weakness. Since human intestinal parasitic infections are high in the study area, mass treatment of people with intestinal protozoans is strongly recommended, especially in the rural areas where the prevalence was very high.

An ex-vivo Human Intestinal Model to Study Entamoeba histolytica Pathogenesis

Amoebiasis (a human intestinal infection affecting 50 million people every year) is caused by the protozoan parasite Entamoeba histolytica. To study the molecular mechanisms underlying human colon invasion by E. histolytica, we have set up an ex vivo human colon model to study the early steps in amoebiasis. Using scanning electron microscopy and histological analyses, we have established that E. histolytica caused the removal of the protective mucus coat during the first two hours of incubation, detached the enterocytes, and then penetrated into the lamina propria by following the crypts of Lieberkühn. Significant cell lysis (determined by the release of lactodehydrogenase) and inflammation (marked by the secretion of pro-inflammatory molecules such as interleukin 1 beta, interferon gamma, interleukin 6, interleukin 8 and tumour necrosis factor) were detected after four hours of incubation. Entamoeba dispar (a closely related non-pathogenic amoeba that also colonizes the human colon) was unable to invade colonic mucosa, lyse cells or induce an inflammatory response. We also examined the behaviour of trophozoites in which genes coding for known virulent factors (such as amoebapores, the Gal/GalNAc lectin and the cysteine protease 5 (CP-A5), which have major roles in cell death, adhesion (to target cells or mucus) and mucus degradation, respectively) were silenced, together with the corresponding tissue responses. Our data revealed that the signalling via the heavy chain Hgl2 or via the light chain Lgl1 of the Gal/GalNAc lectin is not essential to penetrate the human colonic mucosa. In addition, our study demonstrates that E. histolytica silenced for CP-A5 does not penetrate the colonic lamina propria and does not induce the host's pro-inflammatory cytokine secretion.

CD8+ T cells clear human intestinal Cryptosporidium infection through cytotoxic granule release (129.8)

Abstract INTRODUCTION: A key factor in the human adaptive immune response towards Cryptosporidium, associated with resistance to infection, is IFNg. Its function and the cells involved in clearing the parasite from the human intestinal epithelium are unknown. As CD4+ and CD8+ cells produce IFNg after re-stimulation with parasite antigens in vitro we hypothesized that CD8+ cells contribute to control of the infection. METHODS: We studied CD8+ cell cytotoxicity by 51Chromium (51Cr) release assay and fluorescent microscopy. Targets were 51Cr labeled CaCo2 cells. Effectors were CD8+ cells isolated from peripheral blood mononuclear cells of sensitized or naïve volunteers after 6 day culture with IL-2 + IL-15 +/- C. hominis glycoprotein 15 (gp15). RESULTS: Gp15 stimulated CD8+ cells from a sensitized donor matched at HLA-A and HLA-B killed infected cells, with killing increasing with effector numbers, in contrast to CD8+ cells from a naïve donor. In microscopy experiments the same primed CD8+ cells released cytolytic granules into infected cells. CD8+ cells from sensitized donors matched at HLA-A, but not at HLA-B did not kill infected targets as efficiently. HLA-A and HLA-B antibodies inhibited cytotoxicity. CONCLUSION: The data suggest that re-stimulated CD8+ cells from sensitized individuals kill infected cells through release of cytotoxic granules. HLA-B seems to be at least partially required for peptide recognition.

Parasitic intestinal infections in humans between 2006 and 2007

Between 2006-07 faecal specimens of 2.132 subjects (1.508 adults and 624 children) were examined for ova & parasites, using direct and after formalin-ethylacetate concentration microscopy, and permanent specific stains. 380 bubjects (17.8 %) were infected: 313 adults (20.8 %) and 67 children (10.7 %). 331 cases (15.5 %) were infected by pathogens, 275 adults (18.2 %) and 56 children (9.0 %). 389 pathogenic or not pathogenic protozoa (18.2 %) and 60 helminths (2.8 %) were identified, more among adults than children (21.0 % vs 11.5 % and 3.2 % vs 1.8 % respectively).Among protozoa, D. fragilis was in all observed in 145 cases (6.8 %), G. duodenalis in 74 cases (3.5 %), other were very rare.Among helminths nematodes were more frequent than trematodes and cestodes, with S. stercoralis (14 cases) and E. vermicularis (13 cases) the most frequent ones. 2.302 subjects (1.505 adults and 797 children) were examined for microbiological tests because affected by acute or prolonged diarrhoea. 82 cases (3.6 %) of protozoal infections were observed, 70 among adults (4.7 %) and 12 among children (1.5 %). D. fragilis was in all prevalent (2.0 %) in respect of G. duodenalis (1.0 %) or other ones (0.6 %). For S. stercoralis specific investigation, modified Baermann method / larvae colture were performed: 20/189 cases (10.6 %) od strongyloidiasis was diagnosed in adults. For E. vermicularis investigation, scotch test was performer: 43/179 cases (24.0%) of enterobiasis was diagnosed. The Authors underline the application of standard operative procedures for O & P with permanent specific stains in subject affected by enterites too, and the analysis of more specimens for each subjects for good diagnostical performances.