Clathrin Heavy Chain subunits coordinate endo- and exocytic traffic and affect stomatal movement

The current model for vesicular traffic to and from the plasma membrane is accepted but the molecular requirements for this coordination are not well defined. We have identified the has1 mutant, which has a stomatal function defect, as a clathrin heavy chain 1 (CHC1) mutant allele and show that it has a decreased rate of endocytosis and growth defects that are shared with other chc1 mutant alleles. We used chc1 alleles and the related chc2 mutant as tools to investigate the effects clathrin defects have on secretion pathways and plant growth. We show that secretion and endocytosis at the plasma membrane is sensitive to CHC1 and CHC2 function in seedling roots, and that chc mutants have physiological defects in stomatal function and plant growth that have not been previously described. These findings suggest that clathrin supports specific functions of multiple cell types. Stomata movement and gas exchange is altered in chc mutants, indicating clathrin is important for stomatal regulation. The aberrant function of chc mutant stomata is consistent with the growth phenotypes observed under different water and light conditions, which are also similar to those of the secretory SNARE mutant, syp121. The syp121 and chc mutants have impaired endo- and exocytosis compared to wild type, indicating a link between SYP121-dependent secretion and clathrin-dependent endocytosis at the plasma membrane. Our findings provide evidence that clathrin and SYP121 functions are important for the coordination of endo- and exocytosis, and have an impact on stomatal function, gas exchange, and vegetative growth in Arabidopsis

B-cell dysregulation in Crohn's disease is partially restored with infliximab therapy

Background: B-cell depletion can improve a variety of chronic inflammatory diseases, but does not appear beneficial for patients with Crohn's disease. Objective: To elucidate the involvement of B cells in Crohn's disease, we here performed an 'in depth' analysis of intestinal and blood B-cells in this chronic inflammatory disease. Methods: Patients with Crohn's disease were recruited to study B-cell infiltrates in intestinal biopsies (n = 5), serum immunoglobulin levels and the phenotype and molecular characteristics of blood B-cell subsets (n = 21). The effects of infliximab treatment were studied in 9 patients. Results: Granulomatous tissue showed infiltrates of B lymphocytes rather than Ig-secreting plasma cells. Circulating transitional B cells and CD21low B cells were elevated. IgM memory B cells were reduced and natural effector cells showed decreased replication histories and somatic hypermutation (SHM) levels. In contrast, IgG and IgA memory B cells were normally present and their Ig gene transcripts carried increased SHM levels. The numbers of transitional and natural effector cells were normal in patients who responded clinically well to infliximab. Conclusions: B cells in patients with Crohn's disease showed signs of chronic stimulation with localization to granulomatous tissue and increased molecular maturation of IgA and IgG. Therapy with TNFα-blockers restored the defect in IgM memory B-cell generation and normalized transitional B-cell levels, making these subsets candidate markers for treatment monitoring. Together, these results suggest a chronic, aberrant B-cell response in patients with Crohn's disease, which could be targeted with new therapeutics that specifically regulate B-cell function

Nuclear positioning rather than contraction controls ordered rearrangements of immunoglobulin loci

Progenitor-B cells recombine their immunoglobulin (Ig) loci to create unique antigen receptors. Despite a common recombination machinery, the Ig heavy and Ig light chain loci rearrange in a stepwise manner. We studied pre-pro-B cells and Rag-/- progenitor-B cells to determine whether Ig locus contraction or nuclear positioning is decisive for stepwise rearrangements. We found that both Ig loci were contracted in pro-B and pre-B cells. Igh relocated from the nuclear lamina to central domains only at the pro-B cell stage, whereas, Igê remained sequestered at the lamina, and only at the pre-B cell stage located to central nuclear domains. Finally, in vitro induced re-positioning of Ig alleles away from the nuclear periphery increased germline transcription of Ig loci in pre-pro-B cells. Thus, Ig locus contraction juxtaposes genomically distant elements to mediate efficient recombination, however, sequential positioning of Ig loci away from the nuclear periphery determines stage-specific accessibility of Ig loci

Decreased IL7Rα and TdT expression underlie the skewed immunoglobulin repertoire of human B-cell precursors from fetal origin

Newborns are unable to mount antibody responses towards certain antigens. This has been related to the restricted repertoire of immunoglobulin (Ig) genes of their B cells. The mechanisms underlying the restricted fetal Ig gene repertoire are currently unresolved. We here addressed this with detailed molecular and cellular analysis of human precursor-B cells from fetal liver, fetal bone marrow (BM), and pediatric BM. In the absence of selection processes, fetal B-cell progenitors more frequently used proximal V, D and J genes in complete IGH gene rearrangements, despite normal Ig locus contraction. Fewer N-nucleotides were added in IGH gene rearrangements in the context of low TdT and XRCC4 expression. Moreover, fetal progenitor-B cells expressed lower levels of IL7Rα than their pediatric counterparts. Analysis of progenitor-B cells from IL7Rα-deficient patients revealed that TdT expression and N-nucleotides additions in Dh-Jh junctions were dependent on functional IL7Rα. Thus, IL7Rα affects TdT expression, and decreased expression of this receptor underlies at least in part the skewed Ig repertoire formation in fetal B-cell precursors. These new insights provide a better understanding of the formation of adaptive immunity in the developing fetus

Pre-B Cell Receptor Signaling Induces Immunoglobulin κ Locus Accessibility by Functional Redistribution of Enhancer-Mediated Chromatin Interactions

During B cell development, the precursor B cell receptor (pre-BCR) checkpoint is thought to increase immunoglobulin κ light chain (Igκ) locus accessibility to the V(D)J recombinase. Accordingly, pre-B cells lacking the pre-BCR signaling molecules Btk or Slp65 showed reduced germline Vκ transcription. To investigate whether pre-BCR signaling modulates Vκ accessibility through enhancer-mediated Igκ locus topology, we performed chromosome conformation capture and sequencing analyses. These revealed that already in pro-B cells the κ enhancers robustly interact with the ∼3.2 Mb Vκ region and its flanking sequences. Analyses in wild-type, Btk, and Slp65 single- and double-deficient pre-B cells demonstrated that pre-BCR signaling reduces interactions of both enhancers with Igκ locus flanking sequences and increases interactions of the 3′κ enhancer with Vκ genes. Remarkably, pre-BCR signaling does not significantly affect interactions between the intronic enhancer and Vκ genes, which are already robust in pro-B cells. Both enhancers interact most frequently with highly used Vκ genes, which are often marked by transcription factor E2a. We conclude that the κ enhancers interact with the Vκ region already in pro-B cells and that pre-BCR signaling induces accessibility through a functional redistribution of long-range chromatin interactions within the Vκ region, whereby the two enhancers play distinct roles

A Randomized Dietary Intervention to Increase Colonic and Peripheral Blood Short-Chain Fatty Acids Modulates the Blood B- and T-cell Compartments in Healthy Humans

BACKGROUND: Short-chain fatty acids (SCFA) have immune-modulating effects in animal models of disease. However, there is limited evidence that this may occur in humans. OBJECTIVES: This study aimed to determine the effects of increased exposure to SCFA via dietary manipulation on colonic fermentation and adaptive immune cells. METHODS: Twenty healthy, young adults (18-45 years of age) underwent a blinded, randomized, cross-over dietary intervention, consuming a high-SCFA producing diet and matched low-SCFA diet for 21 days with 21-day wash-out in between. SCFA were provided through resistant starch, inulin and apple cider vinegar. Blood and 3-day total fecal output were collected at baseline and at the end of each diet. Gas chromatography was used to measure fecal and plasma SCFA. Flow cytometry was used for peripheral blood immuno-phenotyping. RESULTS: High-SCFA diet was associated with significantly (paired samples Wilcoxon test) higher median [IQR] fecal SCFA concentrations (86.6 [59.0] vs 75.4 [56.2] µmol/g, P = 0.02) and significantly lower median fecal ammonia concentrations (26.2 [14.7] vs 33.4 [18.5] µmol/g, P = 0.04) than the low-SCFA diet. Plasma propionate (9.87 [12.3] vs 4.72 [7.6] µmol/L, P = 0.049) and butyrate (2.85 [1.35] vs 2.02 [1.29] µmol/L, P = 0.03) were significantly higher after high-SCFA diet than after low-SCFA diet. Blood total B cells (184 [112] vs 199 [143] cells/µL, P = 0.04), naive B cells (83 [66] vs 95 [89] cells/µL, P = 0.02), Th1 cells (22 [19] vs 29 [16] cells/µL, P = 0.03) and mucosal-associated invariant T (MAIT) cells (62 [83] vs 69 [114] cells/µL, P = 0.02) were significantly lower after high-SCFA diet than low-SCFA diet. CONCLUSION: Increasing colonic and peripheral blood SCFA has discrete effects on circulating immune cells in healthy humans following 3-week intervention. Further studies, e.g., in patients with inflammatory disease, are necessary to determine if these changes have immunomodulatory effects, whether these are therapeutically beneficial, and whether prolonged intake might be required. Clinical trial registry: Australian New Zealand Clinical trials registry: ACTRN12618001054202.

"It Takes Two To Tango", A Research About The Risks And Control Measures For The Management Of A Performance Contract For Regular Maintenance Of Rail Infrastructure.

A research about the risks and the administrative measures during the management of a performance contract for the regular maintenance of rail infrastructure. In the sector of rail infrastructure output-contracts, in which the contractor is judged on achieved performances, are getting more in use. The assumption is that this will lead to a more efficient and innovative execution of the maintenance, because the parties involved in the contract have less contact and thus need to take more responsibilities for their proper tasks. Unknown however is how to manage such a contract: What are the risks a contracting partner is confronted with during the term of the contract and how could these risks be managed? This article will discuss a method that will provide answers to these questions. The method consists of a matrix of the possible risks and control measures that the management level of both the sourcing partner and the contractor should discuss. These are cooperation risks and not the safety and availability risks. Within HTM, the bus and tram company of The Hague, this method is used to give direction for a new maintenance contract. It was concluded that management based on performances not always means that contracting partner and contractor have less contact but that it resulted in another behavior and cooperation both for the contracting partner as the contracting party.Institute of Transport and Logistics Studies. Faculty of Economics and Business. The University of Sydne

IL-21 contributes to type 2 memory B cell formation

Type 2 memory B cells (mBC2), identified in mice as IgG1+CD23+IL-4Rα+CD38+ B cells, which require IL-4 for genesis, are precursors for IgE plasma cells (PC) and considered key in allergy. While IL-21 is critical for normal mBC differentiation, its involvement in mBC2 formation is unknown. Here, we show that although IL-21R-deficient (IL-21R-/-) mice immunized with ovalbumin generated ovalbumin-specific mBC2, their abundances were lower than in controls one-week post-immunization. This deficit was associated with the accumulation of pre-prememory GC B cells and corresponding deficiencies in pre-memory B cell exit, suggesting a defect in memory differentiation. Germinal centers waned rapidly in IL-21R-/- mice and subsequently mBC2 numbers diminished, and IgG1 PC genesis was suppressed. These data show that IL-21 is required for normal mBC2 formation, arguing against the use of IL-21 as a pan-allergy suppressor.</p

Environmental Outcomes of Reducing Medication Waste by Redispensing Unused Oral Anticancer Drugs

Importance: Medications are associated with substantial environmental outcomes, yet frequently end up being unused by patients. Waste-minimizing interventions, such as redispensing of quality-approved oral anticancer drugs remaining unused by patients at home, could reduce the environmental footprint of cancer treatment. Objectives: To assess the environmental outcomes of redispensing quality-assured oral anticancer drugs and to explore how redispensing could be environmentally optimized. Design, Setting, and Participants: In this quality improvement study, a cradle-to-grave life cycle assessment was performed in the outpatient pharmacy of 4 Dutch hospitals, based on a prospective multicenter trial comprising 1071 patients with a clinical diagnosis of cancer and an active prescription for an oral anticancer drug stored at room temperature from February 1, 2021, to February 1, 2023, with a follow-up of 12 months per patient. Intervention: Participants received prescribed oral anticancer drugs with additional quality-assurance materials (ie, seal bags and time-temperature indicators), so the pharmacy could redispense quality-assured drugs based on authenticity, appearance, remaining shelf life, and/or adequate storage. Main Outcomes and Measures: The estimated environmental outcomes avoided due to waste reduction (ie, production and transport and incineration of redispensed oral anticancer drugs) corrected for outcomes of process burdens (ie, quality assurance materials), quantified in 3 outcome measures: human health damage (disability-adjusted life-years), ecosystems damage (species × year), and climate change (kg of carbon dioxide equivalent [CO2-eq]) per patient per year. Results: A volunteer sample of 1071 patients (median age, 70 years [IQR, 62-75 years]; 622 men [58.1%]) participated in the intervention. Redispensing oral anticancer drugs was initially associated with an environmental burden, mainly because of the high impact of time-temperature indicators. However, when quality-assurance materials were selectively used for temperature-sensitive oral anticancer drugs (ie, maximum storage temperature of 25 °C), redispensing was environmentally beneficial to human health and ecosystems, providing estimated climate benefits of 1.9 kg (95% CI, 1.4-2.6 kg) of CO2-eq per patient per year. Conclusions and Relevance: In this quality improvement study, redispensing unused oral anticancer drugs was found to be a suitable strategy to reduce waste and improve environmental sustainability of cancer treatment after process optimization. Redispensing unused oral anticancer drugs could contribute to sustainability of cancer treatment through reduced costs and environmental outcomes