Human induced pluripotent stem cells (BIONi010-C) generate tight cell monolayers with blood-brain barrier traits and functional expression of large neutral amino acid transporter 1 (SLC7A5).

The barrier properties of the brain capillary endothelium, the blood-brain barrier (BBB) restricts uptake of most small and all large molecule drug compounds to the CNS. There is a need for predictive human in vitro models of the BBB to enable studies of brain drug delivery. Here, we investigated whether human induced pluripotent stem cell (hiPSC) line (BIONi010-C) could be differentiated to brain capillary endothelial- like cells (BCEC) and evaluated their potential use in drug delivery studies. BIONi010-C hIPSCs were differentiated according to established protocols. BCEC monolayers displayed transendothelial electrical resistance (TEER) values of 5,829±354 Ω∙cm2, a Papp,mannitol of 1.09±0.15 ∙ 10-6 cm∙s-1 and a Papp,diazepam of 85.7 ± 5.9 ∙ 10-6 cm ∙s-1. The Pdiazepam/Pmannitol ratio of ~80, indicated a large dynamic passive permeability range. Monolayers maintained their integrity after medium exchange. Claudin-5, Occludin, Zonulae Occludens 1 and VE-Cadherin were expressed at the cell-cell contact zones. Efflux transporters were present at the mRNA level, but functional efflux of substrates was not detected. Transferrin-receptor (TFR), Low density lipoprotein receptor-related protein 1 (LRP1) and Basigin receptors were expressed at the mRNA-level. The presence and localization of TFR and LRP1 were verified at the protein level. A wide range of BBB-expressed solute carriers (SLC's) were detected at the mRNA level. The presence and localization of SLC transporters GLUT1 and LAT1 was verified at the protein level. Functional studies revealed transport of the LAT1 substrate [3H]-L-Leucine and the LRP1 substrate angiopep-2. In conclusion, we have demonstrated that BIONi010-C-derived BCEC monolayers exhibited, BBB properties including barrier tightness and integrity, a high dynamic range, expression of some of the BBB receptor and transporter expression, as well as functional transport of LAT1 and LRP1 substrates. This suggests that BIONi010-C-derived BCEC monolayers may be useful for studying the roles of LAT-1 and LRP1 in brain drug delivery.

How Accurate Are Implant Surgical Guides Produced With Desktop Stereolithographic 3-Dimentional Printers?

PURPOSE: The use of tooth-supported static stereolithographic guides has greatly improved the ability to ideally place implants. This study was designed to determine the accuracy of in office-printed implant surgical guides. MATERIALS AND METHODS: Using 3shape Implant Studio, a treatment plan for implant placement for tooth 8 was developed using a digital intraoral scan from a Trios scanner and cone-beam computed tomography. Ten stereolithographic guides were printed using a Form2 3-dimensional printer. Pre- and post-implant insertion digital scans were used to determine distance and angulation differences in the mesiodistal and faciolingual positions of the implants compared with the planned position. RESULTS: The mean difference in mesiodistal direction at the alveolar crest between planned implants and placed implants was 0.28 mm (range, 0.05 to 0.62 mm) and the difference in the faciolingual direction was 0.49 mm (range, 0.08 to 0.72 mm). The mean mesiodistal angulation deviation was 0.84° (range, 0.08° to 4.48°) and the mean faciolingual angulation deviation was 3.37° (range, 1.12° to 6.43°). CONCLUSIONS: In-office fabricated stereolithographic implant surgical guides show similar accuracy to laboratory- or manufacturer-prepared guides. This technique provides a convenient and cost-effective means of assuring proper implant placement.

A large outbreak of gastrointestinal illness at an open-water swimming event in the River Thames, London.

Open-water swimming is increasingly popular, often in water not considered safe for bathing. Limited evidence exists on the associated health risks. We investigated gastrointestinal illness in 1100 swimmers in a River Thames event in London, UK, to describe the outbreak and identify risk factors. We conducted a retrospective cohort study. Our case definition was swimmers with any: diarrhoea, vomiting, abdominal cramps lasting ⩾48 h, nausea lasting ⩾48 h, with onset within 9 days after the event. We used an online survey to collect information on symptoms, demographics, pre- and post-swim behaviours and open-water experience. We tested associations using robust Poisson regression. We followed up case microbiological results. Survey response was 61%, and attack rate 53% (338 cases). Median incubation period was 34 h and median symptom duration 4 days. Five cases had confirmed microbiological diagnoses (four Giardia, one Cryptosporidium). Wearing a wetsuit [adjusted relative risk (aRR) 6·96, 95% confidence interval (CI) 1·04-46·72] and swallowing water (aRR 1·42, 95% CI 1·03-1·97) were risk factors. Recent river-swimming (aRR 0·78, 95% CI 0·67-0·92) and age >40 years (aRR 0·83, 95% CI 0·70-0·98) were protective. Action to reduce risk of illness in future events is recommended, including clarification of oversight arrangements for future swims to ensure appropriate risk assessment and advice is provided.

Acute Flaccid Paralysis: The New, The Old, and The Preventable.

Acute flaccid paralysis (AFP) has a changing epidemiology with ongoing polio outbreaks and emerging causes such as nonpolio enteroviruses and West Nile virus (WNV). We report a case of AFP from the Horn of Africa that was initially classified as probable polio but subsequently found to be due to WNV.

Profound parental bias associated with chromosome 14 acquired uniparental disomy indicates targeting of an imprinted locus.

Acquired uniparental disomy (aUPD) is a common finding in myeloid malignancies and typically acts to convert a somatically acquired heterozygous mutation to homozygosity. We sought to identify the target of chromosome 14 aUPD (aUPD14), a recurrent abnormality in myeloid neoplasms and population cohorts of elderly individuals. We identified 29 cases with aUPD14q that defined a minimal affected region (MAR) of 11.2 Mb running from 14q32.12 to the telomere. Exome sequencing (n=7) did not identify recurrently mutated genes, but methylation-specific PCR at the imprinted MEG3-DLK1 locus located within the MAR demonstrated loss of maternal chromosome 14 and gain of paternal chromosome 14 (P<0.0001), with the degree of methylation imbalance correlating with the level of aUPD (r=0.76; P=0.0001). The absence of driver gene mutations in the exomes of three individuals with aUPD14q but no known haematological disorder suggests that aUPD14q may be sufficient to drive clonal haemopoiesis. Analysis of cases with both aUPD14q and JAK2 V617F (n=11) indicated that aUPD14q may be an early event in some cases but a late event in others. We conclude that aUPD14q is a recurrent abnormality that targets an imprinted locus and may promote clonal haemopoiesis either as an initiating event or as a secondary change.

A certified plasmid reference material for the standardisation of BCR-ABL1 mRNA quantification by real-time quantitative PCR.

Serial quantification of BCR-ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR-ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 10(6), 1.08±0.11 × 10(5), 1.03±0.10 × 10(4), 1.02±0.09 × 10(3), 1.04±0.10 × 10(2) and 10.0±1.5 copies/µl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR-ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR-ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f).

Managing the risk of iatrogenic transmission of Creutzfeldt-Jakob disease in the UK.

BACKGROUND: With the emergence of bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (CJD) in the UK, there is concern about iatrogenic transmission, and the approach to managing this risk is unique. AIM: To describe and review CJD incident management and the notification of individuals 'at increased risk' as a strategy for reducing iatrogenic transmission. METHODS: A description of iatrogenic CJD transmission, the CJD Incidents Panel's role, the number and nature of CJD incidents reported and the individuals considered 'at increased risk' by mid-2012. FINDINGS: Seventy-seven UK cases of CJD are likely to have resulted from iatrogenic transmission, among recipients of human-derived growth hormone (64 cases), dura mater grafts (eight cases), blood transfusions (four cases) and plasma products (one case). To limit transmission, the Panel reviewed 490 incidents and advised on look-backs, recalls of blood and plasma products, and quarantining and disposing of surgical instruments. Additionally, on Panel advice, around 6000 asymptomatic individuals have been informed they are at increased risk of CJD and have been asked to follow public health precautions. CONCLUSION: The strategy to reduce iatrogenic transmission of CJD has been developed in a context of scientific uncertainty. The rarity of transmission events could indicate that incident-related exposures present negligible transmission risks, or--given the prolonged incubation and subclinical phenotypes of CJD--infections could be yet to occur or have been undetected. Scientific developments, including better estimates of infection prevalence, a screening test, or improvements in decontaminating surgical instruments, may change future risk management.

No evidence of transmission from an acute case of hepatitis A in a foodhandler: follow-up of almost 1,000 potentially exposed individuals, London, United Kingdom, April 2012.

Identification of acute hepatitis A virus (HAV) infection in a foodhandler in a London hotel led to a large incident response. We identified three potentially exposed groups: hotel staff who had regularly consumed food prepared by the case and shared toilet facilities with the case, patients who shared the same hospital ward as the case and hotel guests who consumed food prepared by the case. We arranged post-exposure HAV vaccination for all 83 potentially exposed hotel staff and all 17 patients. We emailed 887 guests advising them to seek medical care if symptomatic, but did not advise vaccination as it was too late to be effective for most guests. Through the International Health Regulations national focal points and the European Union Early warning and response system (EWRS), we communicated the details of the incident to public health agencies and potential risk of HAV transmission to international guests. Potentially exposed hotel staff and guests were asked to complete an online or telephone-administered questionnaire 50 days following possible exposure, to identify any secondary cases. Survey response was low, with 155 responses from guests and 33 from hotel staff. We identified no secondary cases of HAV infection through follow-up.

Early embryonic development, assisted reproductive technologies, and pluripotent stem cell biology in domestic mammals.

Over many decades assisted reproductive technologies, including artificial insemination, embryo transfer, in vitro production (IVP) of embryos, cloning by somatic cell nuclear transfer (SCNT), and stem cell culture, have been developed with the aim of refining breeding strategies for improved production and health in animal husbandry. More recently, biomedical applications of these technologies, in particular, SCNT and stem cell culture, have been pursued in domestic mammals in order to create models for human disease and therapy. The following review focuses on presenting important aspects of pre-implantation development in cattle, pigs, horses, and dogs. Biological aspects and impact of assisted reproductive technologies including IVP, SCNT, and culture of pluripotent stem cells are also addressed.

Antimicrobial susceptibility testing of Clostridium difficile using EUCAST epidemiological cut-off values and disk diffusion correlates.

With the emergence of reduced susceptibility of Clostridium difficile to metronidazole and vancomycin the value of antimicrobial susceptibility testing has increased. The aim of our study was to evaluate disk diffusion for susceptibility testing of C. difficile by comparing disk diffusion results with MICs from gradient tests and to propose zone diameter breakpoint correlates for the EUCAST epidemiological cut-off values (ECOFFs) recently published. We tested 211 clinical isolates of C. difficile, from patients with diarrhoea hospitalized at Aarhus and Odense University Hospitals, Denmark. Furthermore, ten clinical isolates of C. difficile from the Anaerobe Reference Laboratory, University Hospital of Wales, with known reduced susceptibility to either metronidazole or vancomycin, were included. Isolates were tested with Etest gradient strips and disk diffusion towards metronidazole, vancomycin and moxifloxacin on Brucella Blood Agar supplemented with hemin and vitamin K. We found an excellent agreement between inhibition zone diameter and MICs. For each MIC value, the inhibition zones varied from 0 to 8 mm, with 93% of values within 6 mm for metronidazole, 95% of values within 4 mm for vancomycin, and 98% of values within 4 mm for moxifloxacin. With proposed zone diameter breakpoints for metronidazole, vancomycin and moxifloxacin of WT ≥ 23 mm, WT ≥ 19 and WT ≥ 20 mm, respectively, we found no very major errors and only major errors below 2%. In conclusion, we suggest that disk diffusion is an option for antimicrobial susceptibility testing of C. difficile.

Understanding gut-immune interactions in management of acute infectious diarrhoea.

This article discusses the role that immunity plays in the risk of diarrhoea and the potential role for probiotics in the management of acute infectious diarrhoea in older people, including antibiotic-associated diarrhoea and Clostridium difficile-associated diarrhoea.

Directed differentiation of porcine epiblast-derived neural progenitor cells into neurons and glia.

Neural progenitor cells (NPCs) are promising candidates for cell-based therapy of neurodegenerative diseases; however, safety concerns must be addressed through transplantation studies in large animal models, such as the pig. The aim of this study was to derive NPCs from porcine blastocysts and evaluate their in-vitro differentiation potential. Epiblasts were manually isolated from expanded hatched blastocysts and cultured on MEF feeder cells. Outgrowth colonies were passaged to MS5 cells and rosettes were further passaged to Matrigel-coated dishes containing bFGF and EGF. Three NPC lines were established which showed expression of SOX2, NESTIN and VIMENTIN. One line was characterised in more detail, retaining a normal karyotype and proliferating for more than three months in culture. Following differentiation, TUJI was significantly up-regulated in protocol 2 (RA and SHH; 58% positive cells) as were NF and TH. In contrast, MBP was significantly up-regulated in protocol 3 (FGF8 and SHH; 63% positive cells), whereas, GFAP was significantly up-regulated in protocols 1-4 (33%, 25%, 43% and 22%). The present study provides the first report of a porcine blastocyst-derived NPC line capable of differentiating into both neurons and glia, which may be of paramount importance for future transplantation studies in large animal models of neurodegenerative diseases.

A case of confined placental mosaicism with double trisomy associated with stillbirth.

We present a case of stillbirth in which the fetus was well grown and karyotypically normal, but the placenta was morphologically abnormal and had confined placental mosaicism (CPM) for a double trisomy of chromosomes 12 and 15. A compilation of published cases of CPM reveals that whilst approximately 80% of pregnancies progress normally, there is an association with abnormal placental morphology, intrauterine growth restriction, fetal abnormalities and stillbirth. This case highlights the potential adverse effects of CPM and the benefit of placental examination in determining the cause of stillbirth.

University students' notebook computer use: lessons learned using e-diaries to report musculoskeletal discomfort.

The objective of this pilot study was to identify if notebook accessories (ergonomic chair, desktop monitor and notebook riser) combined with a wireless keyboard, mouse and participatory ergonomics training would have the greatest impact on reducing self-reported upper extremity musculoskeletal discomfort in university students. In addition to pre-post computing and health surveys, the Ecological Momentary Assessment was used to capture change in discomfort over time using a personal digital assistant (PDA) as the e-diary. The PDA was programmed with a survey containing 45 questions. Four groups of university students were randomised to either intervention (three external computer accessories) or to control. Participants reported less discomfort with the ergonomic chair and notebook riser based on the pre-post survey data and the e-diary/PDA ANOVA analysis. However, the PDA data, adjusted for the effect of hours per day of computer use, showed no benefit of the chair and limited benefit from the riser. Statement of Relevance:University students' use of notebook computers has increased. This study found evidence of a positive effect of an adjustable chair or notebook riser when combined with ergonomic training on reducing discomfort. Daily notebook computer use of 4 h was confirmed as a risk factor. Without some form of ergonomic intervention, these students are likely to enter the workforce with poor computing habits, which places them on the road to future injuries as technology continues to play a dominant role in their lives.

Aquagenic wrinkling of the palms in an adult cystic fibrosis population.

BACKGROUND: Aquagenic wrinkling of the palms (AWP) is a rare condition characterised by the development of oedema and excessive wrinkling of the palms following exposure to water. It has frequently been associated with cystic fibrosis (CF). Early reports of AWP have only been case reports or small case series; there has only been one reported prevalence study of AWP in a CF population. OBJECTIVE: To determine the incidence and characteristics of AWP in the adult CF population in Northern Ireland. METHODS: 105 CF patients were interviewed. The patients were asked whether they noticed excess wrinkling of the hands when exposed to water. If they answered 'yes', further questions were asked regarding clinical characteristics. The atopic status, CF genotype and drug history were recorded for each patient. Formal testing of 7 patients was carried out. RESULTS: Out of the 105 patients who were interviewed, 43 (41%) described AWP. Of the 43 patients with AWP, 20 were male and 23 were female. There was no association of AWP with genotype, atopy or concomitant drug intake. CONCLUSION: AWP appears to have an equal sex incidence, and the high number of cases in the population studied would suggest that this condition is underreported.

Clinical phenotype of cystic fibrosis patients with the G551D mutation.

BACKGROUND: Data on whether the phenotype of cystic fibrosis (CF) patients with compound heterozygocity for G551D (Gly551Asp) differs from patients with F508del (Phe508del) homozygous mutations is divergent. AIM: We hypothesized that CF patients with the G551D mutation would have less severe disease than F508del homozygotes. DESIGN: We compared the clinical phenotype of adult patients with a G551D mutation with adult patients homozygous for F508del and those with the missense mutation R117H (Arg117His). Compound heterozygotes for the G551D and R117H were analysed separately. METHODS: Data were collected for 101 adult CF patients. Group 1-4 represents in order F508del homozygote patients (n = 61), those with the G551D mutation and a more severe mutation (n = 13), those with R117H mutation and a more severe mutation (n = 23) and also those compound for both the R117H and G551D mutations (n = 4). RESULTS: Our findings have shown that adult patients with the G551D mutation and a second severe mutation have a milder clinical phenotype than F508del homozygous adult patients. Higher FEV(1) and body mass index and less impaired glucose tolerance was demonstrated in the patients with G551D and R117H compared to F508del homozygotes. There was a reduced yearly rate of decline of FEV(1) (P < 0.05), infection with Pseudomonas aeruginosa along with reduced burden of care. Compound heterozygosity for G551D and R117H mutations was associated with normal spirometry, body mass index, no chronic infection and no symptoms. CONCLUSION: Mutations on different chromosomes are not independent of each other for the overall impact on the amount of functional CFTR. This study suggests that patients with the G551D mutation and a second severe mutation have a milder clinical phenotype than F508del homozygous patients, but the phenotype is not as mild as patients with the R117H mutation.

From zygote to implantation: morphological and molecular dynamics during embryo development in the pig.

The increasing focus on the pig as a biomedical model calls for studies which investigate morphological and molecular mechanisms during initial embryonic development in this species. In the pig, the paternal genome is actively demethylated in the zygote, whereas the maternal genome remains methylated. The major genome activation occurs at the four-cell stage, when prominent ribosome-synthesizing nucleoli develop in the blastomeres, allowing for trophectoderm and inner cell mass (ICM) differentiation. Unlike in mice, the pluripotency gene OCT4 is initially expressed in both compartments. The ICM differentiates into epiblast and hypoblast approximately at the time of hatching from the zona pellucida, and subsequently the loss of the Rauber's layer results in an uncovered epiblast establishing the embryonic disc again in contrast to mice. This particular and protracted ICM/epiblast biology may contribute to the lack of success in culturing porcine embryonic stem cells. The embryonic disc subsequently becomes polarized by a posterior thickening, which includes ingression of the first extra-embryonic mesoderm. Thereafter, the primitive streak forms and gastrulation results in formation of the somatic germ layers and germline, i.e. the primordial germ cells. The latter remain pluripotent for a period and may be isolated and cultured as embryonic germ cells in vitro.