Crohn's disease: an in vitro analysis of T lymphocyte function and response to commensal microbes

Inflammatory bowel disease is a chronic, relapsing inflammation of the intestine. Approximately 0.5% of the western world is estimated to suffer from the disease. Crohn’s disease, a type of inflammatory bowel disease, consists of a patchy inflammation, that can occur throughout the entire gastrointestinal tract. Due to the heterogeneous nature of Crohn’s disease, little is known about the mechanisms of disease pathogenesis. Compared to healthy individuals, patients with Crohn’s disease often have elevated levels of inflammatory T lymphocyte subsets, their associated effector cytokines, and higher intestinal permeability. Crohn’s disease has been associated with genetic risk factors, sedentary lifestyles, and a loss of tolerance to the commensal members of the microbiome. In this research, T lymphocytes from non-IBD and Crohn’s disease patient peripheral blood mononuclear cells (PBMCs) were analysed for comparison. Flow cytometry was used to fluorescently label cells and analytes of interest. Visual analytic tools, such as CytoAnalytics’ EarlyBird and viSNE were used to visualise patient variation within, and between groups. It was hypothesised that Crohn’s disease T lymphocytes would have a bias for inflammation-inducing T lymphocyte subsets, and that these cells would have a higher capacity for inflammation, than non-IBD controls. Indeed, inflammatory T lymphocytes were present at higher frequencies in Crohn’s disease patient blood samples. Furthermore, Crohn’s disease patient PBMCs had a higher capacity for proliferation in response to T lymphocyte stimulation. In responses to the commensal bacterium, Faecalibacterium prausnitzii, Crohn’s disease patient PBMC Tregs increased in frequency, whereas CD4+ inflammatory T lymphocyte subsets maintained their frequency. In contrast, non-IBD PBMC Tregs were not influenced by co-culture with F. prausnitzii, and CD4+ inflammatory T lymphocyte subsets decreased in the presence of F. prausnitzii. Together, these data suggest that Crohn’s disease patient PBMCs respond abnormally to commensal bacteria, and this could play a role in disease pathogenesis. Intestinal organoids are derived from patient colonic biopsy stem cells. Organoids provide a unique in vitro model for the observation of intestinal interactions. In this thesis, a 2D intestinal monolayer was developed from the culture of 3D intestinal organoids. 2D monolayers emulate epithelial intestinal barrier cellular organisation. A difficult question to answer in Crohn’s disease research is whether intestinal permeability and inflammation is a cause or consequence of disease. As such, patient organoids and bacteria were integrated into the model stepwise, to analyse the influence each component has on the system, in the absence, or presence, on an active immune presence. Using this model, it was found that monolayers derived from Crohn’s disease patients were more permeable than non-IBD controls. Furthermore, Crohn’s disease patient monolayers had deleterious interactions in co-culture with F. prausnitzii, resulting in reduced epithelial integrity; in contrast to non-IBD patient monolayers which were unaffected. The addition of matched PBMCs into Crohn’s disease patient monolayers exacerbated epithelial degradation in the presence of F. prausnitzii. Taken together, it was found that Crohn’s disease patient PBMCs had greater inflammatory capacity than non-IBD control PBMCs. These data suggest that intestinal cells in patients with Crohn’s disease may have deleterious interactions with commensal bacteria, which could result in increased intestinal permeability. Furthermore, Crohn’s disease patient Treg responses to commensal bacteria suggest that intestinal areas of immune suppression may promote bacteria success, and potential dysbiosis. Translocation of the luminal microbiota into the lamina propria, combined with excessive T lymphocyte inflammatory capacity, could be an initial driving factor for Crohn’s disease pathogenesis. Data in this thesis provide valuable insight into the initiation of epithelial degradation in patients with Crohn’s disease. These data suggest that epithelial degradation may occur in Crohn’s disease patients in response to commensal bacteria, even in the absence of an immune cell influence. With further development, the intestinal organoid monolayer model may provide insight into the mechanisms of epithelial degradation in individual patients. Further, the monolayer model may also provide a tool to optimise individual patient treatments, in vitro

What do we know about demand, use and outcomes in primary care out-of-hours services? A systematic scoping review of international literature

This study was funded by the Scottish Government through the Primary Care Division and Health Improvement Scotland.Objective To synthesise international evidence for demand, use and outcomes of primary care out-of-hours health services (OOHS). Design Systematic scoping review. Data sources CINAHL; Medline; PsyARTICLES; PsycINFO; SocINDEX; and Embase from 1995 to 2019. Study selection English language studies in UK or similar international settings, focused on services in or directly impacting primary care. Results 105 studies included: 54% from mainland Europe/Republic of Ireland; 37% from UK. Most focused on general practitioner-led out-of-hours cooperatives. Evidence for increasing patient demand over time was weak due to data heterogeneity, infrequent reporting of population denominators and little adjustment for population sociodemographics. There was consistent evidence of higher OOHS use in the evening compared with overnight, at weekends and by certain groups (children aged 65, women, those from socioeconomically deprived areas, with chronic diseases or mental health problems). Contact with OOHS was driven by problems perceived as urgent by patients. Respiratory, musculoskeletal, skin and abdominal symptoms were the most common reasons for contact in adults; fever and gastrointestinal symptoms were the most common in the under-5s. Frequent users of daytime services were also frequent OOHS users; difficulty accessing daytime services was also associated with OOHS use. There is some evidence to suggest that OOHS colocated in emergency departments (ED) can reduce demand in EDs. Conclusions Policy changes have impacted on OOHS over the past two decades. While there are generalisable lessons, a lack of comparable data makes it difficult to judge how demand has changed over time. Agreement on collection of OOHS data would allow robust comparisons within and across countries and across new models of care. Future developments in OOHS should also pay more attention to the relationship with daytime primary care and other services.Publisher PDFPeer reviewe

Inhibition of delta-like ligand 4 induces luteal hypervascularization followed by functional and structural luteolysis in the primate ovary

Using specific inhibitors established that angiogenesis in the ovarian follicle and corpus luteum is driven by vascular endothelial growth factor. Recently, it has been demonstrated that the Notch ligand, delta-like ligand 4 (Dll4) negatively regulates vascular endothelial growth factor-mediated vessel sprouting and branching. To investigate the role of Dll4 in regulation of the ovarian vasculature, we administered a neutralizing antibody to Dll4 to marmosets at the periovulatory period. The vasculature was examined on luteal d 3 or d 10: angiogenesis was determined by incorporation of bromodeoxyuridine, staining for CD31 and cell death by staining for activated caspase-3. Ovulatory progesterone rises were monitored to determine effects of treatment on luteal function and time to recover normal cycles in a separate group of animals. Additionally, animals were treated in the follicular or midluteal phase to determine effects of Dll4 inhibition on follicular development and luteal function. Controls were treated with human IgG (Fc). Corpora lutea from marmosets treated during the periovulatory period exhibited increased angiogenesis and increased vascular density on luteal d 3, but plasma progesterone was significantly suppressed. By luteal d 10, corpora lutea in treated ovaries were significantly reduced in size, with involution of luteal cells, increased cell death, and suppressed plasma progesterone concentrations. In contrast, initiation of anti-Dll4 treatment during the midluteal phase produced only a slight suppression of progesterone for the remainder of the cycle. Moreover, Dll4 inhibition had no appreciable effect on follicular development. These results show that Dll4 has a specific and critical role in the development of the normal luteal vasculature

Perinatal germ cell development and differentiation in the male marmoset (Callithrix jacchus):similarities with the human and differences from the rat

STUDY QUESTION: Is perinatal germ cell (GC) differentiation in the marmoset similar to that in the human? SUMMARY ANSWER: In a process comparable with the human, marmoset GC differentiate rapidly after birth, losing OCT4 expression after 5–7 weeks of age during mini-puberty. WHAT IS KNOWN ALREADY: Most of our understanding about perinatal GC development derives from rodents, in which all gonocytes (undifferentiated GC) co-ordinately lose expression of the pluripotency factor OCT4 and stop proliferating in late gestation. Then after birth these differentiated GC migrate to the basal lamina and resume proliferation prior to the onset of spermatogenesis. In humans, fetal GC differentiation occurs gradually and asynchronously and OCT4(+) GC persist into perinatal life. Failure to switch off OCT4 in GC perinatally can lead to development of carcinoma in situ (CIS), the precursor of testicular germ cell cancer (TGCC), for which there is no animal model. Marmosets show similarities to the human, but systematic evaluation of perinatal GC development in this species is lacking. Similarity, especially for loss of OCT4 expression, would support use of the marmoset as a model for the human and for studying CIS origins. STUDY DESIGN, SIZE AND DURATION: Testis tissues were obtained from marmosets (n = 4–10 per age) at 12–17 weeks' gestation and post-natal weeks 0.5, 2.5, 5–7, 14 and 22 weeks, humans at 15–18 weeks' gestation (n = 5) and 4–5 weeks of age (n = 4) and rats at embryonic day 21.5 (e21.5) (n = 3) and post-natal days 4, 6 and 8 (n = 4 each). PARTICIPANTS/MATERIALS, SETTING AND METHODS: Testis sections from fetal and post-natal marmosets, humans and rats were collected and immunostained for OCT4 and VASA to identify undifferentiated and differentiated GC, respectively, and for Ki67, to identify proliferating GC. Stereological quantification of GC numbers, differentiation (% OCT4(+) GC) and proliferation were performed in perinatal marmosets and humans. Quantification of GC position within seminiferous cords was performed in marmosets, humans and rats. MAIN RESULTS AND ROLE OF CHANCE: The total GC number increased 17-fold from birth to 22 post-natal weeks in marmosets; OCT4(+) and VASA(+) GC proliferated equally in late gestation and early post-natal life. The percentage of OCT4(+) GC fell from 54% in late fetal life to <0.5% at 2.5 weeks of age and none were detected after 5–7 weeks in marmosets. In humans, the percentage of OCT4(+) GC also declined markedly during the equivalent period. In marmosets, GC had begun migrating to the base of seminiferous cords at ∼22 weeks of age, after the loss of GC OCT4 expression. LIMITATIONS, REASONS FOR CAUTION: There is considerable individual variation between marmosets. Although GC development in marmosets and humans was similar, there are differences with respect to proliferation during fetal life. The number of human samples was limited. WIDER IMPLICATIONS OF THE FINDINGS: The similarities in testicular GC differentiation between marmosets and humans during the perinatal period, and their differences from rodents, suggest that the marmoset may be a useful model for studying the origins of CIS, with relevance for the study of TGCC. STUDY FUNDING/COMPETING INTERESTS: This work was supported by Grant G33253 from the Medical Research Council, UK. No external funding was sought and there are no competing interests

An immortalised mesenchymal stem cell line maintains mechano-responsive behaviour and can be used as a reporter of substrate stiffness

The mechanical environment can influence cell behaviour, including changes to transcriptional and proteomic regulation, morphology and, in the case of stem cells, commitment to lineage. However, current tools for characterizing substrates’ mechanical properties, such as atomic force microscopy (AFM), often do not fully recapitulate the length and time scales over which cells ‘feel’ substrates. Here, we show that an immortalised, clonal line of human mesenchymal stem cells (MSCs) maintains the responsiveness to substrate mechanics observed in primary cells, and can be used as a reporter of stiffness. MSCs were cultured on soft and stiff polyacrylamide hydrogels. In both primary and immortalised MSCs, stiffer substrates promoted increased cell spreading, expression of lamin-A/C and translocation of mechano-sensitive proteins YAP1 and MKL1 to the nucleus. Stiffness was also found to regulate transcriptional markers of lineage. A GFP-YAP/RFP-H2B reporter construct was designed and virally delivered to the immortalised MSCs for in situ detection of substrate stiffness. MSCs with stable expression of the reporter showed GFP-YAP to be colocalised with nuclear RFP-H2B on stiff substrates, enabling development of a cellular reporter of substrate stiffness. This will facilitate mechanical characterisation of new materials developed for applications in tissue engineering and regenerative medicine

Antarctic ice rises and rumples: Their properties and significance for ice-sheet dynamics and evolution

Locally grounded features in ice shelves, called ice rises and rumples, play a key role buttressing discharge from the Antarctic Ice Sheet and regulating its contribution to sea level. Ice rises typically rise several hundreds of meters above the surrounding ice shelf; shelf flow is diverted around them. On the other hand, shelf ice flows across ice rumples, which typically rise only a few tens of meters above the ice shelf. Ice rises contain rich histories of deglaciation and climate that extend back over timescales ranging from a few millennia to beyond the last glacial maximum. Numerical model results have shown that the buttressing effects of ice rises and rumples are significant, but details of processes and how they evolve remain poorly understood. Fundamental information about the conditions and processes that cause transitions between floating ice shelves, ice rises and ice rumples is needed in order to assess their impact on ice-sheet behavior. Targeted high-resolution observational data are needed to evaluate and improve prognostic numerical models and parameterizations of the effects of small-scale pinning points on grounding-zone dynamics

Aggressive Regimens for Multidrug-Resistant Tuberculosis Decrease All-Cause Mortality

Rationale: A better understanding of the composition of optimal treatment regimens for multidrug-resistant tuberculosis (MDR-TB) is essential for expanding universal access to effective treatment and for developing new therapies for MDR-TB. Analysis of observational data may inform the definition of an optimized regimen. Objectives: This study assessed the impact of an aggressive regimen–one containing at least five likely effective drugs, including a fluoroquinolone and injectable–on treatment outcomes in a large MDR-TB patient cohort. Methods: This was a retrospective cohort study of patients treated in a national outpatient program in Peru between 1999 and 2002. We examined the association between receiving an aggressive regimen and the rate of death. Measurements and Main Results: In total, 669 patients were treated with individualized regimens for laboratory-confirmed MDR-TB. Isolates were resistant to a mean of 5.4 (SD 1.7) drugs. Cure or completion was achieved in 66.1% (442) of patients; death occurred in 20.8% (139). Patients who received an aggressive regimen were less likely to die (crude hazard ratio [HR]: 0.62; 95% CI: 0.44,0.89), compared to those who did not receive such a regimen. This association held in analyses adjusted for comorbidities and indicators of severity (adjusted HR: 0.63; 95% CI: 0.43,0.93). Conclusions: The aggressive regimen is a robust predictor of MDR-TB treatment outcome. TB policy makers and program directors should consider this standard as they design and implement regimens for patients with drug-resistant disease. Furthermore, the aggressive regimen should be considered the standard background regimen when designing randomized trials of treatment for drug-resistant TB

Roadmap on Photovoltaic Absorber Materials for Sustainable Energy Conversion

Photovoltaics (PVs) are a critical technology for curbing growing levels of anthropogenic greenhouse gas emissions, and meeting increases in future demand for low-carbon electricity. In order to fulfil ambitions for net-zero carbon dioxide equivalent (CO2eq) emissions worldwide, the global cumulative capacity of solar PVs must increase by an order of magnitude from 0.9 TWp in 2021 to 8.5 TWp by 2050 according to the International Renewable Energy Agency, which is considered to be a highly conservative estimate. In 2020, the Henry Royce Institute brought together the UK PV community to discuss the critical technological and infrastructure challenges that need to be overcome to address the vast challenges in accelerating PV deployment. Herein, we examine the key developments in the global community, especially the progress made in the field since this earlier roadmap, bringing together experts primarily from the UK across the breadth of the photovoltaics community. The focus is both on the challenges in improving the efficiency, stability and levelized cost of electricity of current technologies for utility-scale PVs, as well as the fundamental questions in novel technologies that can have a significant impact on emerging markets, such as indoor PVs, space PVs, and agrivoltaics. We discuss challenges in advanced metrology and computational tools, as well as the growing synergies between PVs and solar fuels, and offer a perspective on the environmental sustainability of the PV industry. Through this roadmap, we emphasize promising pathways forward in both the short- and long-term, and for communities working on technologies across a range of maturity levels to learn from each other.Comment: 160 pages, 21 figure

Clinical outcomes and response to treatment of patients receiving topical treatments for pyoderma gangrenosum: a prospective cohort study

Background: pyoderma gangrenosum (PG) is an uncommon dermatosis with a limited evidence base for treatment. Objective: to estimate the effectiveness of topical therapies in the treatment of PG. Methods: prospective cohort study of UK secondary care patients with a clinical diagnosis of PG suitable for topical treatment (recruited July 2009 to June 2012). Participants received topical therapy following normal clinical practice (mainly Class I-III topical corticosteroids, tacrolimus 0.03% or 0.1%). Primary outcome: speed of healing at 6 weeks. Secondary outcomes: proportion healed by 6 months; time to healing; global assessment; inflammation; pain; quality-of-life; treatment failure and recurrence. Results: Sixty-six patients (22 to 85 years) were enrolled. Clobetasol propionate 0.05% was the most commonly prescribed therapy. Overall, 28/66 (43.8%) of ulcers healed by 6 months. Median time-to-healing was 145 days (95% CI: 96 days, ∞). Initial ulcer size was a significant predictor of time-to-healing (hazard ratio 0.94 (0.88;80 1.00); p = 0.043). Four patients (15%) had a recurrence. Limitations: No randomised comparator Conclusion: Topical therapy is potentially an effective first-line treatment for PG that avoids possible side effects associated with systemic therapy. It remains unclear whether more severe disease will respond adequately to topical therapy alone