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

Variation in Structure and Process of Care in Traumatic Brain Injury: Provider Profiles of European Neurotrauma Centers Participating in the CENTER-TBI Study.

INTRODUCTION: The strength of evidence underpinning care and treatment recommendations in traumatic brain injury (TBI) is low. Comparative effectiveness research (CER) has been proposed as a framework to provide evidence for optimal care for TBI patients. The first step in CER is to map the existing variation. The aim of current study is to quantify variation in general structural and process characteristics among centers participating in the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study. METHODS: We designed a set of 11 provider profiling questionnaires with 321 questions about various aspects of TBI care, chosen based on literature and expert opinion. After pilot testing, questionnaires were disseminated to 71 centers from 20 countries participating in the CENTER-TBI study. Reliability of questionnaires was estimated by calculating a concordance rate among 5% duplicate questions. RESULTS: All 71 centers completed the questionnaires. Median concordance rate among duplicate questions was 0.85. The majority of centers were academic hospitals (n = 65, 92%), designated as a level I trauma center (n = 48, 68%) and situated in an urban location (n = 70, 99%). The availability of facilities for neuro-trauma care varied across centers; e.g. 40 (57%) had a dedicated neuro-intensive care unit (ICU), 36 (51%) had an in-hospital rehabilitation unit and the organization of the ICU was closed in 64% (n = 45) of the centers. In addition, we found wide variation in processes of care, such as the ICU admission policy and intracranial pressure monitoring policy among centers. CONCLUSION: Even among high-volume, specialized neurotrauma centers there is substantial variation in structures and processes of TBI care. This variation provides an opportunity to study effectiveness of specific aspects of TBI care and to identify best practices with CER approaches

Variation in general supportive and preventive intensive care management of traumatic brain injury: a survey in 66 neurotrauma centers participating in the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study

Abstract Background General supportive and preventive measures in the intensive care management of traumatic brain injury (TBI) aim to prevent or limit secondary brain injury and optimize recovery. The aim of this survey was to assess and quantify variation in perceptions on intensive care unit (ICU) management of patients with TBI in European neurotrauma centers. Methods We performed a survey as part of the Collaborative European NeuroTrauma Effectiveness Research in Traumatic Brain Injury (CENTER-TBI) study. We analyzed 23 questions focused on: 1) circulatory and respiratory management; 2) fever control; 3) use of corticosteroids; 4) nutrition and glucose management; and 5) seizure prophylaxis and treatment. Results The survey was completed predominantly by intensivists (n = 33, 50%) and neurosurgeons (n = 23, 35%) from 66 centers (97% response rate). The most common cerebral perfusion pressure (CPP) target was > 60 mmHg (n = 39, 60%) and/or an individualized target (n = 25, 38%). To support CPP, crystalloid fluid loading (n = 60, 91%) was generally preferred over albumin (n = 15, 23%), and vasopressors (n = 63, 96%) over inotropes (n = 29, 44%). The most commonly reported target of partial pressure of carbon dioxide in arterial blood (PaCO2) was 36–40 mmHg (4.8–5.3 kPa) in case of controlled intracranial pressure (ICP) < 20 mmHg (n = 45, 69%) and PaCO2 target of 30–35 mmHg (4–4.7 kPa) in case of raised ICP (n = 40, 62%). Almost all respondents indicated to generally treat fever (n = 65, 98%) with paracetamol (n = 61, 92%) and/or external cooling (n = 49, 74%). Conventional glucose management (n = 43, 66%) was preferred over tight glycemic control (n = 18, 28%). More than half of the respondents indicated to aim for full caloric replacement within 7 days (n = 43, 66%) using enteral nutrition (n = 60, 92%). Indications for and duration of seizure prophylaxis varied, and levetiracetam was mostly reported as the agent of choice for both seizure prophylaxis (n = 32, 49%) and treatment (n = 40, 61%). Conclusions Practice preferences vary substantially regarding general supportive and preventive measures in TBI patients at ICUs of European neurotrauma centers. These results provide an opportunity for future comparative effectiveness research, since a more evidence-based uniformity in good practices in general ICU management could have a major impact on TBI outcome

Mucosal melanomas of the head and neck: State of the art and current controversies

Mucosal melanomas of the head and neck (sinonasal and oral cavity) account for 1% of neoplasms, 4% of all melanomas and over 50% of all mucosal melanomas. They have a high metastatic potential. Five-year overall survival does not exceed 30%. Diagnosis may be difficult and includes adequate immunohistochemical staining. Risk factors, presentation and molecular biology are different from those of cutaneous melanomas. The mainstay of treatment is surgery and postoperative radiotherapy. Endoscopic surgery should be evaluated prospectively. Neck dissection is recommended for N0 oral cavity melanomas, while it can generally be omitted for sinonasal melanomas. Inoperable tumors can be treated with exclusive radiotherapy. Molecular guidance for metastatic cases is a relevant option despite low level of evidence, based on the rarity of disease and low response rates to chemotherapy. c-KIT inhibitors and immunotherapy appear promising

Newly visualized fibrillar collagen scaffolds dictate Entamoeba histolytica invasion route in the human colon

International audienceThe extracellular matrix (ECM) and its role in the outcome of infectious diseases have been poorly investigated. In this study, we determined the impact of the collagen fibres architecture on the invasive process of the enteric parasite Entamoeba histolytica. The behaviour of E. histolytica wild-type and silenced for the cysteine protease A5 (CP-A5) were compared on a three-dimensional collagen matrix and within human colon fragments for fibrillar collagen cleavage and migration. The interstitial collagen fibres within the connective tissue of the human colon, visualized by multiphoton and second harmonic generation signals imaging, presented a dense scaffold at the subepithelial level and a loose meshwork within the chorion. To penetrate the tissue, E. histolytica migrated on the dense scaffold that remained intact, reached the crypt of Lieberkhün, migrated along and then disorganized the loose scaffold to escape into the mucosa. Interestingly, in vitro, CP-A5 was not required for collagenase activity and migration through the matrix but was necessary within the tissue environment for collagen meshwork remodelling and subsequent invasion. The data point out that further step of invasion relay with ECM destruction that requires human components induced or activated in the presence of CP-A5

Variants des carcinomes épidermoïdes dans les voies aérodigestives supérieures (VADS), implications pour le diagnostic et la prise en charge, selon les référentiels du REFCOR

International audienceParmi les 20 000 nouveaux cas de cancers ORL, tête et cou et des voies aérodigestives (VADS) enFrance, les carcinomes épidermoïdes des VADS représentent environ 90 % des cas. Parmi ceux-ci,les variants des carcinomes épidermoïdes dits conventionnels représentent entre 5 % et 10 % descas. Un recensement précis est difficile car ils ne sont pas actuellement colligés de façonsystématique. L'anamnèse et le terrain sont rarement différents de ceux des carcinomes épi-dermoïdes conventionnels. Ces variants des carcinomes épidermoïdes peuvent pourtant êtresujets à des erreurs diagnostiques, voire à des erreurs thérapeutiques, du fait de confusions avecdes sarcomes, des tumeurs glandulaires, voire des tumeurs bénignes. Certaines précautionsd'analyse (par biopsies profondes par exemple) doivent être connues pour ne pas omettre leurcomposante la plus agressive en cas de tumeurs avec contingents variés. L'immunohistochimie et certaines analyses moléculaires spécialisées peuvent, de plus, aider au diagnostic. Une relecturediagnostique paraît indispensable pour certains de ces variants. Par ailleurs, certains variants sontradiorésistants et à l'inverse, d'autres radiosensibles. Dans les autres cas, des compléments d'analysesmoléculaires peuvent redresser certaines erreurs diagnostiques et permettre des traitements adé-quats. Une actualisation des référentiels REFCOR 2008 a été réalisée au vu de la littérature inter-nationale et de la nouvelle classification OMS/IARC (Organisation mondiale de la santé/Internationalagency for research on cancer) 2017 pour les sept principaux variants de carcinomes épidermoïdesdes VADS, les carcinomes verruqueux, acantholytiques (ou adénoïdes), basaloïdes, papillaires, à cel-lules fusiformes (improprement nommés sarcomatoïdes, adénosquameux et lymphoépithéliaux). Leséléments diagnostiques et thérapeutiques différenciants y sont rapportés

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