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

Cell cytotoxicity during interaction between human colonic explants and <i>E. histolytica</i> or <i>E. dispar</i>.

<p>Mean LDH concentrations (IU/L) released into the supernatant of the organotypic culture after incubation with <i>E. histolytica</i> WT or <i>E. dispar</i> or in the absence of amoeba (control) from 1 to 7 hours. Data are from 8 individual experiments. <b>*</b> indicates a significant difference between WT and control (<i>p</i><0.03) and between WT and <i>E. dispar</i> (<i>p</i><0.05). <b>#</b> indicates a significant difference between WT and control (<i>p</i><0.001) and between WT and <i>E. dispar</i> (<i>p</i><0.02)</p

Migration through the lamina propria of <i>E. histolytica</i> sub-strains impaired in virulent functions.

<p>Representative images from three individual experiments are shown. Histological examination of colonic tissue sections after seven hours of incubation, with HGL2, G3, RBV and RB8. Transversal tissue slices were stained with haematoxylin-eosin. Trophozoites were immunostained with antibodies against the Gal/GalNAc lectin. Experiments with HGL2, G3 and RBV revealed that trophozoites were able to invade the mucosa, as described for the WT. In contrast, RB8 parasites were unable to penetrate deeper into the lamina propria and were blocked at the surface of the mucosa, although they were still able to disorganize and detach cells from the upper side of the mucosa.</p

Interaction between Entamoeba and the lumen surface of the human colonic explants.

<p><b>A</b>. Analyse by histology of the mucus layer at the surface of Human colonic fragments incubated for seven hours without <i>Entamoeba</i> (left panel) and with <i>E. histolytica</i> (right panel). The mucus layer covering the epithelium at the surface was observable after seven hours of organotypic culture but not in the presence of <i>E. histolytica</i>. <b>B</b>. Scanning electron micrographs of the luminal surface of the human colonic explants incubated with <i>E. histolytica</i> or <i>E. dispar</i>. Representative images from three individual experiments are shown. (a) <i>E. histolytica</i> trophozoites adhering to the mucus layer at time 0; (b) 2 hours after incubation, the mucus layer had been degraded by <i>E. histolytica</i> and the regular mucosal architecture of the colonic epithelium was visible. Holes corresponded to the crypts of Lieberkühn and abundant aggregates were seen in the interglandular regions. (c) The aggregates were composed of human cells and trophozoites, as seen in an enlargement of this region (d) After 4 hours, the epithelium was damaged and (e) <i>E. histolytica</i> trophozoites began to penetrate into the tissue (f) After 4 hours, <i>E. dispar</i> trophozoites were still adhering to the mucus but had not degraded it and (g) had not evoked the recruitment of cells to the interglandular region, as shown after manually scraping the mucus after SEM fixation procedure of the sample.</p

<i>E. histolytica</i>-induced secretion of pro-inflammatory cytokines.

<p>Histogram showing mean ± SD concentrations (pg/ml) of individual analytes (IL-1β, IL-6, IL-8, IFN-γ and TNF) secreted from 8 human colonic explants incubated with WT, <i>E. dispar</i> or without amoeba (control) after 4 and 7 hours of incubation, as measured on a Luminex100 system. Levels of secreted pro-inflammatory cytokines (IL-1β, IL-6, IL-8, IFN-γ and TNF) were significantly higher at 4 hours (<b>*</b><i>p</i><0.05) and 7 hours (<b># </b><i>p</i><0.03) in the explants incubated with WT, in comparison with both those secreted by explants incubated with <i>E. dispar</i> and the amoeba-free control.</p

Pro-inflammatory cytokines secretion induced in the <i>ex-vivo</i> human colonic model by sub-strains of <i>E. histolytica</i>.

<p>Mean concentrations (pg/ml) of IL-1β, IL-8, IFN-γ and TNF secreted after 4 hours (grey bars) and 7 hours (black bars) of incubation of HGL2, NEO, RBV, G3, WT and RB8 trophozoites with 3 individual human colonic explants. NEO and HGL2 parasites induced significantly higher levels of pro-inflammatory cytokines IL-1β, IL-6, IL-8, IFN-γ and TNF in the explants incubated for 4 hours (<i>p</i><0.05) and 7 hours (<i>p</i><0.02), compared with the control in the absence of amoeba. RBV, G3 and WT strains induced significantly higher levels of pro-inflammatory cytokines [IL-1β (<i>p</i><0.05,<0.01 and <0.01 respectively) IL-8 (<0.04,<0.01 and <0.01 respectively), IFN-γ(<0.009,<0.003 and <0,01 respectively) and TNF (<0.02,<0.02 and <0.02 respectively)] in the explants incubated for 4 hours and IL-8 (<0.009, <0.001 and <0.01 respectively), IFN-γ (<0.002,<0.0004 and <0.01 respectively) and TNF (<0.008,<0.008 and <0.02 respectively)] at 7 hours, compared with the control in the absence of amoeba. WT secreted IL1β (0.04/0.03), IL-8 (0.001/0.001), IFN-γ (0.001/0.002) and TNF (0.008/0.01) at 4 and 7 hours respectively, compared with RB8.</p

Cell cytotoxicity during interplay between <i>E. histolytica</i> trophozoites affected for virulent factors and human colonic explants.

<p>Mean LDH concentrations (IU/L, from 3 individual experiments) released after incubation of human colonic explants with HGL2, NEO, RBV, G3, WT and RB8 trophozoites and in the absence of amoeba (control) for 4 hours (grey bars) and 7 hours (black bars). <b>*</b> indicates a significant difference between HGL2 and control (<i>P</i><0.01), NEO and control (<i>P</i><0.03) and WT and control (<i>P</i><0.05). <b>#</b> indicates a significant difference between HGL2 and control (<i>p</i><0.008), NEO and control (<i>p</i><0.01), RBV and control (<i>p</i><0.01), G3 and control (<i>p</i><0.009), WT and control (<i>p</i><0.007), RBV and RB8 (<i>p</i><0.03), G3 and RB8 (<i>p</i><0.01) and WT and RB8 (<i>p</i><0.01)</p

A National French Consensus on Gene List for the Diagnosis of Charcot–Marie–Tooth Disease and Related Disorders Using Next-Generation Sequencing

International audienceNext generation sequencing (NGS) is strategically used for genetic diagnosis in patients with Charcot–Marie–Tooth disease (CMT) and related disorders called non-syndromic inherited peripheral neuropathies (NSIPN) in this paper. With over 100 different CMT-associated genes involved and ongoing discoveries, an important interlaboratory diversity of gene panels exists at national and international levels. Here, we present the work of the French National Network for Rare Neuromuscular Diseases (FILNEMUS) genetic diagnosis section which coordinates the seven French diagnosis laboratories using NGS for peripheral neuropathies. This work aimed to establish a unique, simple and accurate gene classification based on literature evidence. In NSIPN, three subgroups were usually distinguished: (1) HMSN, Hereditary Motor Sensory Neuropathy, (2) dHMN, distal Hereditary Motor Neuropathy, and (3) HSAN, Hereditary Sensory Autonomic Neuropathy. First, we reported ClinGen evaluation, and second, for the genes not evaluated yet by ClinGen, we classified them as “definitive” if reported in at least two clinical publications and associated with one report of functional evidence, or “limited” otherwise. In total, we report a unique consensus gene list for NSIPN including the three subgroups with 93 genes definitive and 34 limited, which is a good rate for our gene’s panel for molecular diagnostic use

Mechanisms for variable expressivity of inherited SCN1A mutations causing Dravet syndrome

International audienceBackground. Mutations in SCN1A can cause Genetic Epilepsy with Febrile Seizures Plus (GEFS+, inherited missense mutations) or Dravet syndrome (DS, de novo mutations of all types). Although the mutational spectra are distinct, these disorders share major features and 10% of DS patients have an inherited SCN1A mutation. Objectives and patients. We studied 19 selected families with at least one DS patients to describe the mechanisms accounting for inherited SCN1A mutations in DS. The mutation identified in the DS probands was searched in available parents and relatives and quantified in the blood cells of the transmitting parent using quantitative allele-specific assays. Results. Mosaicism in the blood cells of the transmitting parent was demonstrated in 12 cases and suspected in another case. The proportion of mutated allele in the blood varied from 0.04% to 85%. In the 6 remaining families, six novel missense mutations were associated with autosomal dominant variable GEFS+ phenotypes including DS as the more severe clinical picture. Conclusion. Our results indicate that mosaicism is found in at least 7% of families with at least one DS patient and that it accounts for 68 % (13/19) of inherited mutations associated with DS. On the contrary, in the remaining cases (6/19, 32%), the patients were part of multiplex GEFS+ families and seemed to represent the extreme end of the GEFS+ clinical spectrum. In this latter case, additional genetic or environmental factors likely modulate the severity of the expression of the mutation