Gastrointestinal (GI) infections in sheep have significant implications for animal health, welfare and productivity, as well as being a source of zoonotic pathogens. Interactions between pathogens ...and epithelial cells at the mucosal surface play a key role in determining the outcome of GI infections; however, the inaccessibility of the GI tract
in vivo
significantly limits the ability to study such interactions in detail. We therefore developed ovine epithelial organoids representing physiologically important gastric and intestinal sites of infection, specifically the abomasum (analogous to the stomach in monogastrics) and ileum. We show that both abomasal and ileal organoids form self-organising three-dimensional structures with a single epithelial layer and a central lumen that are stable in culture over serial passage. We performed RNA-seq analysis on abomasal and ileal tissue from multiple animals and on organoids across multiple passages and show the transcript profile of both abomasal and ileal organoids cultured under identical conditions are reflective of the tissue from which they were derived and that the transcript profile in organoids is stable over at least five serial passages. In addition, we demonstrate that the organoids can be successfully cryopreserved and resuscitated, allowing long-term storage of organoid lines, thereby reducing the number of animals required as a source of tissue. We also report the first published observations of a helminth infecting gastric and intestinal organoids by challenge with the sheep parasitic nematode
Teladorsagia circumcincta
, demonstrating the utility of these organoids for pathogen co-culture experiments. Finally, the polarity in the abomasal and ileal organoids can be inverted to make the apical surface directly accessible to pathogens or their products, here shown by infection of apical-out organoids with the zoonotic enteric bacterial pathogen
Salmonella enterica
serovar Typhimurium. In summary, we report a simple and reliable
in vitro
culture system for generation and maintenance of small ruminant intestinal and gastric organoids. In line with 3Rs principals, use of such organoids will reduce and replace animals in host-pathogen research.
Anti-Hu antibodies (Hu-Ab) and anti-CV2/CRMP5 antibodies (CV2/CRMP5-Ab) have been identified in association with paraneoplastic neurological disorders. However, it is not clear whether these ...antibodies are associated with specific neurological symptoms or are only markers of anti-cancer immune reaction.
To address this question, 37 patients with CV2/CRMP5-Ab and 324 patients with Hu-Ab were compared.
Whereas the age and sex ratio were the same between the two groups, the distribution of neurological symptoms was not. Patients with CV2/CRMP5-Ab presented more frequently cerebellar ataxia, chorea, uveo/retinal symptoms and myasthenic syndrome (Lambert-Eaton myasthenic syndrome LEMS or myasthenia gravis). They also had a better Rankin score. In contrast, dysautonomia, brainstem encephalitis and peripheral neuropathy were more frequent in patients with Hu-Ab. Limbic encephalitis occurred similarly in both groups. Small-cell lung cancer was the most frequently associated tumour in both groups of patients, while malignant thymoma was observed only in patients with CV2/CRMP5-Ab. In particular, patients with CV2/CRMP5-Ab and thymoma developed myasthenic syndrome more frequently, while patients with SCLC developed neuropathies more frequently. Chorea and myasthenic syndrome were only seen in patients with CV2/CRMP5-Ab. The median survival time was significantly longer in patients with CV2/CRMP5-Ab, and this effect was not dependent on the type of tumour.
The data demonstrate that in patients with paraneoplastic neurological syndromes, the neurological symptoms and survival vary with both the type of associated onco-neural antibody and the type of tumour.
Few publications on primary Trigeminal Neuralgia treated by Micro-Vascular Decompression (MVD) report large series, with long-term follow-up, using Kaplan-Meier (K-M) analysis. None was specifically ...directed to the comparative study of MVD effectiveness on Trigeminal Neuralgia with typical (i.e., with paroxysmal pain only) and atypical features (i.e., with association of a permanent background of pain).
The authors report a series of 362 patients having clearcut vascular compression and treated with pure MVD - i.e., without any additional cut or coagulation of the adjacent root fibers. Follow-up was 1 to 18 y (8 y on average, with a median of 7.2 y). Results were considered overall, then separately for patients with typical (237 (65.5%)) and atypical (125 (34.5%)) clinical presentation.
One year after operation, (294 (81.2%) of patients were totally-free - of paroxysmal pain, and also of permanent background pain - and not needing any medication) 13 (3.6%) still had a background of pain but without the need for medication which 55 patients (15.2%), treatment had failed. At latest review (8 y on average) the corresponding rates were 80, 4.9 and 15.1%, respectively. Kaplan-Meier analysis estimated the probability of total cure at 15 y to be 73.4%. There was no difference in the cure rate between patients with typical and atypical features at one year: 81 and 81.16%, respectively. The probability of cure at 15 y was identical for the two clinical presentations.
Pure MVD offers patients affected by Trigeminal Neuralgia due to vascular compression a long-lasting cure in three-fourths of the cases. Both typical and atypical presentations respond well to MVD, view in contrast to the classical view that an atypical presentation has an adverse effect on outcome after surgery.
Abstract Aim In digestive cancers, the prognostic significance of intraperitoneal free cancer cells remains unclear (IPCC). The main objective of this study was to assess the prognostic significance ...of IPCC in colorectal and gastric adenocarcinoma. The secondary objectives were to evaluate the predictive significance of IPCC for the development of peritoneal carcinomatosis (PC) and to evaluate the prevalence of synchronous PC and IPCC. Methods This was a prospective multicentre study. All patients undergoing surgery for a digestive tract cancer had peritoneal cytology taken. Patients with gastric and colorectal cancer with no residual tumour after surgery and no evidence of PC were followed-up for 2 years. The primary end point was overall survival. Results Between 2002 and 2007, 1364 patients were enrolled and 956 were followed-up over 2 years. Prevalence of IPCC was 5.7% in colon cancer, 0.6% in rectal cancer and 19.5% in gastric cancer. The overall 2-year survival rate for patients with IPCC was 34.7% versus 86.8% for patients with negative cytology ( p < 0.0001). By multivariate analysis, IPCC was not an independent prognostic factor. No relationship between cytology and recurrence was found. Conclusion The presence of IPCC was not an independent prognostic and didn't add any additional prognostic information to the usual prognostic factors related to the tumour (pTNM and differentiation). Moreover the presence of IPCC detected with this method didn't appear to predict development of PC. Peritoneal cytology using conventional staining doesn't seem to be a useful tool for the staging of colorectal and gastric cancers.
Objective To assess the safety and efficiency of steam vein sclerosis (SVS) of the great saphenous vein (GSV) in a multicentre open prospective cohort study. Design 75 consecutive adult patients with ...GSV reflux, CEAP C2–C5 and vein diameter 4–13 mm. Methods Patients treated using an SVS™ generator delivering homogenous pulses of superheated steam were followed up at 8 days and 1, 3, 6 and 12 months (clinical, duplex ultrasound, quality of life QoL with SF12). Results 88 veins were treated in 75 patients. At 6 months, 72/75 (96%) veins were obliterated (95% CI: 89–99) and Kaplan–Meier analysis found an obliteration rate of 96.1% at 12 months. QoL increased at 6 months for both the physical and mental components ( p = 0.049 and p < 0.001 respectively). SVS was well tolerated: no major complications were reported. Adverse events occurred mainly at day 8 and incidents amounted to ecchymosis ( n = 60) and pain ( n = 7). Conclusions SVS achieved an obliteration rate similar to that of other thermal ablation techniques. It was well tolerated with minimal post-operative pain.
A total on-line analysis of a target protein from a plasma sample was made using a selective immunoextraction step coupled on-line to an immobilized enzymatic reactor (IMER) for the protein digestion ...followed by LC–MS/MS analysis. For the development of this device, cytochrome
c was chosen as model protein due to its well-known sequence. An immunosorbent (IS) based on the covalent immobilization of anti-cytochrome
c antibodies on a solid support was made and an immunoextraction procedure was carefully developed to assess a selective extraction of the target protein from plasma. For the first time, IS was easily coupled on-line with a laboratory-made IMER based on pepsin. The whole on-line device (IS-IMER-LC-MS/MS) allowed the quantification of cytochrome
c from 8.5
pmol to 1.7
nmol in buffer medium. Finally, this device was applied to the analysis of only 85
pmol of cytochrome
c from plasma with a RSD value lower than 10% (
n
=
3).
Two molecularly imprinted polymers (MIPs), prepared in dichloromethane with terbutylazine and ametryn as template molecules, were evaluated for the selective extraction of triazines from complex ...matrices. Various parameters affecting extraction recoveries on MIPs were studied in order to obtain an optimized extraction procedure allowing to reduce non-specific interactions. In order to test the selectivity of the MIPs, the same procedure was applied to the extraction of compounds possessing the same polarity and size as the triazines. By means of molecular modeling, the effects of the electric charge distribution and of the size of the molecules on the retention mechanism were studied. The value of capacity for terbutylazine MIP was also measured. At last, the high selectivity resulting from the use of MIPs was clearly demonstrated by their applications to the clean-up of grape juice and soil extracts spiked with triazines. In addition, the soil extract was cleaned-up by immunoextraction allowing the comparison of both approaches in terms of selectivity.
Direct, selective solid-phase extraction of triazines from aqueous samples is presented using a molecularly imprinted polymer (MIP) made with terbutylazine as template molecule. After optimization of ...the steps of the procedure, 14 triazines including degradation products were studied and satisfactory extraction recoveries were obtained except for thiotriazines. By comparing results obtained with the terbutylazine MIP and a similar non-imprinted polymer, it was determined that retention was achieved via specific interactions except for hydroxyterbutylazine. Selectivity of the extraction procedure was also verified by applying the MIP for the extraction of phenylureas that were not retained on it. The effects of the charge distribution and of molecular volume of the triazines (obtained by molecular modeling) on the selectivity of interactions between the analytes and the MIP were studied. However, when the optimized procedure was applied to real samples, low extraction recoveries were obtained due to strong matrix effects: ion-exchange occurs between the carboxylate groups of the MIP and the ionic species of the sample, that prevents subsequent specific interactions. By introducing an acid wash step, the procedure was successfully applied for the class-selective extraction of triazines from industrial effluent and surface water samples. Finally, increased extraction recoveries were achieved for the polar degradation products of triazines by using a mixed-phase composed of a polymeric sorbent and the MIP.
Traditional
investigation of fungal infection and new antifungal therapies in mouse models is usually carried out using post mortem methodologies. However, biomedical imaging techniques focusing on ...non-invasive techniques using bioluminescent and fluorescent proteins have become valuable tools. These new techniques address ethical concerns as they allow reduction in the number of animals required to evaluate new antifungal therapies. They also allow better understanding of the growth and spread of the pathogen during infection. In this review, we concentrate on imaging technologies using different fungal reporter proteins. We discuss the advantages and limitations of these different reporters and compare the efficacy of bioluminescent and fluorescent proteins for fungal research.
An in-line procedure for protein analysis using a trypsin-based immobilized enzymatic reactor (IMER) coupled to LC–MS/MS has been developed. Various IMERs were synthesized and characterized by ...estimating the digestion yield of a pattern peptide in UV detection. Laboratory-made IMERs were optimized by studying the effect of different parameters as the nature of the functionalized immobilization support (silica, agarose), the amount of immobilized trypsin and the binding density. The potential of the laboratory-made IMERs were compared with a batch digestion and with a commercial trypsin-based IMER. The laboratory-made IMER based on CNBr-activated Sepharose showed the best performances in terms of digestion yields, digestion time, price and repeatability (RSD<4%). Cytochrome c was then digested on this IMER and used in-line with LC–MS. The target protein was easily recognized by the Mascot database until 17pmol injected.
PDF
