In addition to well-characterized CD34+ hematopoietic stem and progenitor cells (HSPCs), the human hematopoietic stem cell (HSC) hierarchy contains a rare CD34− population with severe combined ...immunodeficiency-repopulating capacity. However, little is known about the molecular characteristics of these CD34− cells or their relationship to the CD34+ populations. Here, we show that the self-renewing Lin−CD34−CD38−CD93hi population contains cells that not only function as HSCs, but can also be placed above the CD34+ populations in the hematopoietic hierarchy. These cells have an active Notch pathway, in which signaling through Delta4 is crucial for maintenance of the primitive state, and combined signals from Jagged1 and TGF-β are important in controlling its quiescence. They are also refractory to proliferative signals and show a repressed canonical Wnt pathway, in part regulated by Notch. Overall, therefore, CD34− cells represent an immature and quiescent human HSC population maintained through a distinctive network of cellular signaling interactions.
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•Human CD34− HSCs have self-renewal and repopulating capacities•Cells in this population are immature and quiescent•Distinctive cellular and molecular features include active Notch and TGF-β pathways•Repressive canonical Wnt signaling is in part regulated by Notch
Cellular and molecular analysis of human CD34− HSCs puts them at the top of the pile in the hematopoietic hierarchy, above the previously characterized CD34+ populations.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The concentration of free cytosolic Ca2+ and the voltage across the plasma membrane are major determinants of cell function. Ca2+-permeable non-selective cationic channels are known to regulate these ...parameters, but understanding of these channels remains inadequate. Here we focus on transient receptor potential canonical 4 and 5 proteins (TRPC4 and TRPC5), which assemble as homomers or heteromerize with TRPC1 to form Ca2+-permeable non-selective cationic channels in many mammalian cell types. Multiple roles have been suggested, including in epilepsy, innate fear, pain, and cardiac remodeling, but limitations in tools to probe these channels have restricted progress. A key question is whether we can overcome these limitations and develop tools that are high-quality, reliable, easy to use, and readily accessible for all investigators. Here, through chemical synthesis and studies of native and overexpressed channels by Ca2+ and patch-clamp assays, we describe compound 31, a remarkable small-molecule inhibitor of TRPC1/4/5 channels. Its potency ranged from 9 to 1300 pm, depending on the TRPC1/4/5 subtype and activation mechanism. Other channel types investigated were unaffected, including TRPC3, TRPC6, TRPV1, TRPV4, TRPA1, TRPM2, TRPM8, and store-operated Ca2+ entry mediated by Orai1. These findings suggest identification of an important experimental tool compound, which has much higher potency for inhibiting TRPC1/4/5 channels than previously reported agents, impressive specificity, and graded subtype selectivity within the TRPC1/4/5 channel family. The compound should greatly facilitate future studies of these ion channels. We suggest naming this TRPC1/4/5-inhibitory compound Pico145.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
B cells produce alloantibodies and activate alloreactive T cells, negatively affecting kidney transplant survival. By contrast, regulatory B cells are associated with transplant tolerance. ...Immunotherapies are needed that inhibit B-cell effector function, including antibody secretion, while sparing regulators and minimising infection risk. B lymphocyte stimulator (BLyS) is a cytokine that promotes B-cell activation and has not previously been targeted in kidney transplant recipients. We aimed to determine the safety and activity of an anti-BLyS antibody, belimumab, in addition to standard-of-care immunosuppression in adult kidney transplant recipients. We used an experimental medicine study design with multiple secondary and exploratory endpoints to gain further insight into the effect of belimumab on the generation of de-novo IgG and on the regulatory B-cell compartment.
We undertook a double-blind, randomised, placebo-controlled phase 2 trial of belimumab, in addition to standard-of-care immunosuppression (basiliximab, mycophenolate mofetil, tacrolimus, and prednisolone) at two centres, Addenbrooke's Hospital, Cambridge, UK, and Guy's and St Thomas' Hospital, London, UK. Participants were eligible if they were aged 18–75 years and receiving a kidney transplant and were planned to receive standard-of-care immunosuppression. Participants were randomly assigned (1:1) to receive either intravenous belimumab 10 mg per kg bodyweight or placebo, given at day 0, 14, and 28, and then every 4 weeks for a total of seven infusions. The co-primary endpoints were safety and change in the concentration of naive B cells from baseline to week 24, both of which were analysed in all patients who received a transplant and at least one dose of drug or placebo (the modified intention-to-treat mITT population). This trial has been completed and is registered with ClinicalTrials.gov, NCT01536379, and EudraCT, 2011–006215–56.
Between Sept 13, 2013, and Feb 8, 2015, of 303 patients assessed for eligibility, 28 kidney transplant recipients were randomly assigned to receive belimumab (n=14) or placebo (n=14). 25 patients (12 86% patients assigned to the belimumab group and 13 93% patients assigned to the placebo group) received a transplant and were included in the mITT population. We observed similar proportions of adverse events in the belimumab and placebo groups, including serious infections (one 8% of 12 in the belimumab group and five 38% of 13 in the placebo group during the 6-month on-treatment phase; and none in the belimumab group and two 15% in the placebo group during the 6-month follow-up). In the on-treatment phase, one patient in the placebo group died because of fatal myocardial infarction and acute cardiac failure. The co-primary endpoint of a reduction in naive B cells from baseline to week 24 was not met. Treatment with belimumab did not significantly reduce the number of naive B cells from baseline to week 24 (adjusted mean difference between the belimumab and placebo treatment groups −34·4 cells per μL, 95% CI −109·5 to 40·7).
Belimumab might be a useful adjunct to standard-of-care immunosuppression in renal transplantation, with no major increased risk of infection and potential beneficial effects on humoral alloimmunity.
GlaxoSmithKline.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Normal organogenesis requires co-ordinate development and interaction of multiple cell types, and is seemingly governed by tissue specific factors. Lymphoid organogenesis during embryonic life is ...dependent on molecules the temporal expression of which is tightly regulated. During this process, haematopoietic 'inducer' cells interact with stromal 'organizer' cells, giving rise to the lymphoid organ primordia. Here we show that the haematopoietic cells in the gut exhibit a random pattern of motility before aggregation into the primordia of Peyer's patches, a major component of the gut-associated lymphoid tissue. We further show that a CD45+CD4-CD3-Il7R -c-Kit+CD11c+ haematopoietic population expressing lymphotoxin has an important role in the formation of Peyer's patches. A subset of these cells expresses the receptor tyrosine kinase RET, which is essential for mammalian enteric nervous system formation. We demonstrate that RET signalling is also crucial for Peyer's patch formation. Functional genetic analysis revealed that Gfra3-deficiency results in impairment of Peyer's patch development, suggesting that the signalling axis RET/GFR 3/ARTN is involved in this process. To support this hypothesis, we show that the RET ligand ARTN is a strong attractant of gut haematopoietic cells, inducing the formation of ectopic Peyer's patch-like structures. Our work strongly suggests that the RET signalling pathway, by regulating the development of both the nervous and lymphoid system in the gut, has a key role in the molecular mechanisms that orchestrate intestine organogenesis.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Concerns about university students’ mental wellbeing have been rising and various studies have attempted to unpick the factors that could impact their wellbeing. This focus group study explored the ...impact that virtual learning environments (VLEs) may have on undergraduate students’ mental wellbeing. Forty-four undergraduate students from on-campus courses at three UK universities participated in 12 focus groups in 2020. Using reflexive thematic analysis with an inductive approach, three themes were generated: (1) lecturer VLE-use supports or undermines students’ mental wellbeing; (2) access to the VLE affects students’ productivity, academic performance, and mental wellbeing; and (3) students’ mindset towards the VLE impacts their studies and mental wellbeing. The dominant pattern across the data set was that the way lecturers used the VLE impacted students’ motivation, ability to think clearly about their studies, and could provoke strong emotions. We discuss how the mechanisms described in self-determination theory and the technology acceptance model might explain how the VLE could impact student mental wellbeing.
Predation often has consistent effects on prey behavior and morphology, but whether the physiological mechanisms underlying these effects show similarly consistent patterns across different ...populations remains an open question. In vertebrates, predation risk activates the hypothalamic-pituitary-adrenal (HPA) axis, and there is growing evidence that activation of the maternal HPA axis can have intergenerational consequences via, for example, maternally-derived steroids in eggs. Here, we investigated how predation risk affects a suite of maternally-derived steroids in threespine stickleback eggs across nine Alaskan lakes that vary in whether predatory trout are absent, native, or have been stocked within the last 25 years. Using liquid chromatography coupled with mass spectroscopy (LC-MS/MS), we detected 20 steroids within unfertilized eggs. Factor analysis suggests that steroids covary within and across steroid classes (i.e. glucocorticoids, progestogens, sex steroids), emphasizing the modularity and interconnectedness of the endocrine response. Surprisingly, egg steroid profiles were not significantly associated with predator regime, although they were more variable when predators were absent compared to when predators were present, with either native or stocked trout. Despite being the most abundant steroid, cortisol was not consistently associated with predation regime. Thus, while predators can affect steroids in adults, including mothers, the link between maternal stress and embryonic development is more complex than a simple one-to-one relationship between the population-level predation risk experienced by mothers and the steroids mothers transfer to their eggs.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
The prevalence of mental distress among young adults, including those at university, has increased. In this context, learning analytics, students’ digital trace data, are increasingly being used to ...understand student mental health. In line with calls for more research on learning analytics from student perspectives, as part of a broader focus group study, 44 undergraduate students from three United Kingdom universities were invited to consider how they felt about having a digital footprint on their virtual learning environment (VLE). Two main themes were constructed using reflexive thematic analysis. First, students’ responses depended on the perceived threat to their privacy and identity. Some students were indifferent if no threat was perceived, but expressed unease if there was. Second, some students expressed personal preference for autonomy over use of their VLE data. Two uses identified were for non-judgmental personalized support, and using aggregated data to improve student learning. These themes suggest how the use of educational digital data can, under some circumstances, impact wellbeing negatively. The students’ perspectives garnered from the focus groups could have implications for policy and practice concerning privacy and surveillance, the possibility for misuse or misinterpretation of data, and informed consent. This small study supports the importance of partnering with students to develop and implement guidance for how VLE learning analytics data are used and interpreted by students and staff, including lecturers, to protect and enhance student mental wellbeing.
Thymus organogenesis requires coordinated interactions of multiple cell types, including neural crest (NC) cells, to orchestrate the formation, separation, and subsequent migration of the developing ...thymus from the third pharyngeal pouch to the thoracic cavity. The molecular mechanisms driving these processes are unclear; however, NC-derived mesenchyme has been shown to play an important role. Here, we show that, in the absence of ephrin-B2 expression on thymic NC-derived mesenchyme, the thymus remains in the cervical area instead of migrating into the thoracic cavity. Analysis of individual NC-derived thymic mesenchymal cells shows that, in the absence of ephrin-B2, their motility is impaired as a result of defective EphB receptor signaling. This implies a NC-derived cell-specific role of EphB–ephrin-B2 interactions in the collective migration of the thymic rudiment during organogenesis.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Although data support foregoing preoperative antibiotics for outpatient, soft-tissue procedures, there is a paucity of evidence regarding antibiotics for implant-based hand procedures. The purpose of ...this investigation was to assess early postoperative infectious concerns for patients undergoing implant-based hand surgery, regardless of preoperative antibiotic use.
A retrospective cohort analysis was performed consisting of all patients undergoing implant-based hand procedures between January 2015 and October 2021. Primary outcomes included antibiotic prescription or reoperation for infection within 90 days of surgery. Demographics (age, gender, body mass index, diabetes, and smoking status) and hand surgery procedure type were recorded. To account for differences in baseline characteristics between patients who did and did not receive preoperative antibiotics, covariate balancing was performed with subsequent weighted logistic regression models constructed to estimate the effect of no receipt of preoperative antibiotics on the need for postoperative antibiotics. In a separate logistic regression analysis, patients’ baseline characteristics were evaluated together as predictors of postoperative antibiotic prescription.
One thousand eight hundred sixty-two unique procedures were reviewed with 1,394 meeting criteria. Two hundred thirty-six patients (16.9%) were not prescribed preoperative antibiotics. Overall, 54 (3.87%) and 69 (4.95%) patients received antibiotics within 30 and 90 days of surgery, respectively. One patient (0.07%) underwent reoperation. There were no differences in the rates of 30- and 90-day postoperative antibiotic prescriptions between the two groups. After covariant balancing of risk factors, patients not prescribed preoperative antibiotics did not display significantly higher odds of requiring postoperative antibiotics at 30 or 90 days. Logistic regression models showed male gender, temporary Kirschner wire fixation, and elevated body mass index were associated with increased postoperative antibiotics at 30 and 90 days.
For implant-based hand procedures, there was no increased risk in postoperative antibiotic prescription or reoperation for patients who did not receive preoperative antibiotics.
Therapeutic III.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Lymph node (LN) development depends on prenatal interactions occurring between LN inducer and LN organizer cells. We have distinguished defects in LN formation due to failure in embryonic development ...(aly/aly) from defects in postnatal maturation ($II2r\gamma^{-/-}Rag2^{-/-}$). Both mutant strains form normal primordial LNs with differing fate. In aly/aly mice, the LN primordium dissipates irreversibly late in gestation; in contrast,$II2r\gamma^{-/-}Rag2^{-/-}$LN anlage persists for a week after birth but disperses subsequently, a process reversible by neonatal transfer of WT IL7r⁺ TCR⁺ T or natural killer (NK) cells, suggesting a role for IL7/IL7r interactions. Thus, we reveal a unique stage of postnatal LN development during which mature lymphocytes and IL7/IL7r interactions may play an important role.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
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