Tristan A Cogan

Clinical transplantation of a tissue-engineered airway

BACKGROUND The loss of a normal airway is devastating. Attempts to replace large airways have met with serious problems. Prerequisites for a tissue-engineered replacement are a suitable matrix, cells, ideal mechanical properties, and the absence of antigenicity. We aimed to bioengineer tubular tracheal matrices, using a tissue-engineering protocol, and to assess the application of this technology in a patient with end-stage airway disease. METHODS We removed cells and MHC antigens from a human donor trachea, which was then readily colonised by epithelial cells and mesenchymal stem-cell-derived chondrocytes that had been cultured from cells taken from the recipient (a 30-year old woman with end-stage bronchomalacia). This graft was then used to replace the recipients left main bronchus. FINDINGS The graft immediately provided the recipient with a functional airway, improved her quality of life, and had a normal appearance and mechanical properties at 4 months. The patient had no anti-donor antibodies and was not on immunosuppressive drugs. INTERPRETATION The results show that we can produce a cellular, tissue-engineered airway with mechanical properties that allow normal functioning, and which is free from the risks of rejection. The findings suggest that autologous cells combined with appropriate biomaterials might provide successful treatment for patients with serious clinical disorders.

Stem-cell-based, tissue engineered tracheal replacement in a child: A 2-year follow-up study

BACKGROUND Stem-cell-based, tissue engineered transplants might offer new therapeutic options for patients, including children, with failing organs. The reported replacement of an adult airway using stem cells on a biological scaffold with good results at 6 months supports this view. We describe the case of a child who received a stem-cell-based tracheal replacement and report findings after 2 years of follow-up. METHODS A 12-year-old boy was born with long-segment congenital tracheal stenosis and pulmonary sling. His airway had been maintained by metal stents, but, after failure, a cadaveric donor tracheal scaffold was decellularised. After a short course of granulocyte colony stimulating factor, bone marrow mesenchymal stem cells were retrieved preoperatively and seeded onto the scaffold, with patches of autologous epithelium. Topical human recombinant erythropoietin was applied to encourage angiogenesis, and transforming growth factor β to support chondrogenesis. Intravenous human recombinant erythropoietin was continued postoperatively. Outcomes were survival, morbidity, endoscopic appearance, cytology and proteomics of brushings, and peripheral blood counts. FINDINGS The graft revascularised within 1 week after surgery. A strong neutrophil response was noted locally for the first 8 weeks after surgery, which generated luminal DNA neutrophil extracellular traps. Cytological evidence of restoration of the epithelium was not evident until 1 year. The graft did not have biomechanical strength focally until 18 months, but the patient has not needed any medical intervention since then. 18 months after surgery, he had a normal chest CT scan and ventilation-perfusion scan and had grown 11 cm in height since the operation. At 2 years follow-up, he had a functional airway and had returned to school. INTERPRETATION Follow-up of the first paediatric, stem-cell-based, tissue-engineered transplant shows potential for this technology but also highlights the need for further research. FUNDING Great Ormond Street Hospital NHS Trust, The Royal Free Hampstead NHS Trust, University College Hospital NHS Foundation Trust, and Region of Tuscany.

The rise and fall of Salmonella Enteritidis in the UK

After rising in the early 1980s, the number of recorded human cases of Salmonella enterica subsp. enterica in the UK has fallen in the last 5 years, with a particular decline in cases of infection with serovar Enteritidis. This decline has been concomitant with the introduction of vaccination of egg-laying hens against serovar Enteritidis. It is likely that other factors such as improved biosecurity in egg-laying flocks, a build-up of immunity in other animals and the rise in the number of livestock infections with host-adapted serovars of Salmonella have also played a part in this decline. Although human Salmonella cases are currently at their lowest level since 1987, it is important to remember that the reasons for the dominance of Enteritidis in human infection are poorly understood and it is possible that other serovars could share similar properties and the eradication of Enteritidis may leave a niche for them to fill.

Achieving hygiene in the domestic kitchen: the effectiveness of commonly used cleaning procedures

Aims: To quantify the transmission of Salmonella and Campylobacter to hands, cloths, and hand‐ and food‐contact surfaces during the preparation of raw poultry in domestic kitchens, and to examine the impact on numbers of these bacteria of detergent‐based cleaning alone, or in conjunction with thorough rising.

Norepinephrine increases the pathogenic potential of Campylobacter jejuni

Background:Campylobacter jejuni can cause a spectrum of diseases in humans, ranging from enteritis and diarrhoea to severe inflammation, profuse bloody diarrhoea and chronic relapsing infection. Norepinephrine (NE) levels in the intestine increase under conditions of stress and trauma, and are thought to result in spill over of NE into the intestinal lumen. NE is known to stimulate the growth of a range of bacterial species, and to increase the pathogenicity of Escherichia coli. Aim: To determine the effects of NE on the pathogenic potential of C jejuni in a model system. Methods:C jejuni was grown in iron-replete and iron-limited media in the presence and absence of 100 μM NE. Several virulence-associated characteristics, including motility and cell invasion, were measured. Results: When C jejuni was grown in iron-limited media in the presence of NE, growth rate, motility and invasion of cultured epithelial cells were increased compared with cultures grown in the absence of NE. Bacteria exposed to NE during growth also caused greater subsequent disruption of cultured epithelial cell monolayers, inducing widespread breakdown of tight junctions. Conclusion: Exposure to NE causes an increase in the virulence-associated properties of Campylobacter. Stress and concomitant infection could therefore be contributory factors to the variable presentation of this disease.

Extensive Microbial and Functional Diversity within the Chicken Cecal Microbiome

Chickens are major source of food and protein worldwide. Feed conversion and the health of chickens relies on the largely unexplored complex microbial community that inhabits the chicken gut, including the ceca. We have carried out deep microbial community profiling of the microbiota in twenty cecal samples via 16S rRNA gene sequences and an in-depth metagenomics analysis of a single cecal microbiota. We recovered 699 phylotypes, over half of which appear to represent previously unknown species. We obtained 648,251 environmental gene tags (EGTs), the majority of which represent new species. These were binned into over two-dozen draft genomes, which included Campylobacter jejuni and Helicobacter pullorum. We found numerous polysaccharide- and oligosaccharide-degrading enzymes encoding within the metagenome, some of which appeared to be part of polysaccharide utilization systems with genetic evidence for the co-ordination of polysaccharide degradation with sugar transport and utilization. The cecal metagenome encodes several fermentation pathways leading to the production of short-chain fatty acids, including some with novel features. We found a dozen uptake hydrogenases encoded in the metagenome and speculate that these provide major hydrogen sinks within this microbial community and might explain the high abundance of several genera within this microbiome, including Campylobacter, Helicobacter and Megamonas.

A double-chamber rotating bioreactor for the development of tissue-engineered hollow organs: From concept to clinical trial

Cell and tissue engineering are now being translated into clinical organ replacement, offering alternatives to fight morbidity, organ shortages and ethico-social problems associated with allotransplantation. Central to the recent first successful use of stem cells to create an organ replacement in man was our development of a bioreactor environment. Critical design features were the abilities to drive the growth of two different cell types, to support 3D maturation, to maintain biomechanical and biological properties and to provide appropriate hydrodynamic stimuli and adequate mass transport. An analytical model was developed and applied to predict oxygen profiles in the bioreactor-cultured organ construct and in the culture media, comparing representative culture configurations and operating conditions. Autologous respiratory epithelial cells and mesenchymal stem cells (BMSCs, then differentiated into chondrocytes) were isolated, characterized and expanded. Both cell types were seeded and cultured onto a decellularized human donor tracheal matrix within the bioreactor. One year post-operatively, graft and patient are healthy, and biopsies confirm angiogenesis, viable epithelial cells and chondrocytes. Our rotating double-chamber bioreactor permits the efficient repopulation of a decellularized human matrix, a concept that can be applied clinically, as demonstrated by the successful tracheal transplantation.

Growth of Salmonella enteritidis in artificially contaminated eggs: the effects of inoculum size and suspending media

Growth profiles of two isolates of Salmonella enteritidis phage type (PT) 4 inoculated into either the albumen of whole shell eggs or into separated albumen were found to be markedly affected by the size of the inoculum and the composition of the medium used to suspend the cells prior to inoculation. Using our model with an inoculum of two cells, multiplication of the Salmonella was not seen in 93% of eggs held at 20 degrees C for 8 days. In approximately 7% of eggs, however, growth occurred during the 8 days of storage. If the inoculum equaled or exceeded 25 cells per egg when eggs were subsequently stored at 20 degrees C, or 250 cells per egg when eggs were stored at 30 degrees C, high levels of growth of Salmonella in the egg occurred significantly more frequently than when the inoculum was two cells. High levels of growth were also seen more frequently if the inoculum was suspended in buffered peptone water or maximal recovery diluent rather than in phosphate buffered saline. Growth of Salmonella in separated albumen occurred very infrequently (1.1% of samples) at low inoculum levels and did not become significant until the inoculum was 250 cells or greater. Growth in the albumen was unaffected by the composition of the suspending medium. Provided that the inoculum was approximately 2 cells per egg and the bacteria were suspended in PBS, observed growth profiles of S. enteritidis inoculated into the albumen of whole eggs resembled those in naturally contaminated eggs.

High-Throughput Sequencing of 16S rRNA Gene Amplicons: Effects of Extraction Procedure, Primer Length and Annealing Temperature

The analysis of 16S-rDNA sequences to assess the bacterial community composition of a sample is a widely used technique that has increased with the advent of high throughput sequencing. Although considerable effort has been devoted to identifying the most informative region of the 16S gene and the optimal informatics procedures to process the data, little attention has been paid to the PCR step, in particular annealing temperature and primer length. To address this, amplicons derived from 16S-rDNA were generated from chicken caecal content DNA using different annealing temperatures, primers and different DNA extraction procedures. The amplicons were pyrosequenced to determine the optimal protocols for capture of maximum bacterial diversity from a chicken caecal sample. Even at very low annealing temperatures there was little effect on the community structure, although the abundance of some OTUs such as Bifidobacterium increased. Using shorter primers did not reveal any novel OTUs but did change the community profile obtained. Mechanical disruption of the sample by bead beating had a significant effect on the results obtained, as did repeated freezing and thawing. In conclusion, existing primers and standard annealing temperatures captured as much diversity as lower annealing temperatures and shorter primers.

Prevalence, antibiotic resistance and RAPD typing of Campylobacter species isolated from ducks, their rearing and processing environments in Penang, Malaysia

We report for the first time on the prevalence, antibiotic resistance and RAPD types of Campylobacter species in ducks and duck related environmental samples in Malaysia. Samples were examined by enrichment in Bolton Broth followed by plating onto modified Charcoal Cefoperazone Deoxycholate agar (mCCDA) and/or plating directly onto mCCDA. A total of 643 samples were screened, and the prevalence of Campylobacter spp. in samples from different sources ranged from 0% to 85%. The method of isolation had a significant (P<0.05) effect on the isolation rate. One hundred and sixteen Campylobacter isolates, comprising of 94 Campylobacter jejuni, 19 Campylobacter coli and three Campylobacter lari, were examined for their sensitivity to 13 antibiotics. Majority of the C. jejuni isolates were resistant to cephalothin (99%), tetracycline (96%), suphamethoxazole/trimethoprim (96%), and very few were resistant to gentamicin (5%), chloramphenicol (7%) and erythromycin (1%). All C. coli isolates were resistant to cephalothin, nalidixic acid, norfloxacin and tetracycline but susceptible to chloramphenicol, erythromycin and gentamicin. The three C. lari isolates were resistant to all the antibiotics tested except chloramphenicol and gentamicin (1/3 and 2/3 susceptible, respectively). Genetic diversity of Campylobacter isolates were determined using random amplification of polymorphic DNA (RAPD). C. jejuni and C. coli isolates belong to fifty-eight and twelve RAPD types, respectively.