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The murine anti-human P2X7 receptor monoclonal antibody (mAb) (clone L4) has been used to study the expression and function of the P2X7 receptor on primary leukocytes, keratinocytes, osteoblasts and neuronal cells, as well as various cell lines. This antibody has also been used to characterize polymorphic variants and isoforms of the P2RX7 gene and P2X7 site-directed mutations, and to identify molecules coassociated with P2X7 in the plasma membrane. This chapter describes the maintenance and cryopreservation of the L4 hybridoma cell line, as well as the generation of tissue culture supernatant containing the anti-human P2X7 mAb, and its subsequent purification by Protein A chromatography and conjugation to DyLight™ 488. Moreover, this chapter describes flow cytometric assays to assess the blocking activity and binding of the anti-human P2X7 mAb against P2X7 on human RPMI 8226 multiple myeloma cells.

Humanized mouse models of graft-versus-host disease (GVHD), where human immune cells are injected into immune deficient mice, are well established and provide opportunities to investigate pathways involved in GVHD development. This chapter provides an overview of human immune cell isolation, injection of these cells into immune deficient mice, monitoring of mice for signs of GVHD, and assessment of human cell engraftment using flow cytometry. Further, this chapter focuses on the P2X7 signaling pathway involved in GVHD, and describes a strategy to block the P2X7 receptor and examine the effect of this on GVHD development.

This article uses Feminist New Materialist theoretical perspectives on time and temporality to critically explore the social work practice of life story work, designed to help children who are or have been in public care, or are adopted, assemble a coherent narrative of their care journey, adverse experiences and identity. Interview data presented here was produced with 17 participants (foster carers, adoptive parents and social work professionals in care or adoption teams) who were part of a project which sought to understand how aspects of life story work could be transformed into everyday care conversations between children and the adults who care for them (see www.difficultconversations.info ). Our analysis highlights the way participants understood and constructed temporal elements of children’s experiences and identities: their pasts, presents and futures, in the context of having conversations about care with them. Through the discussion, we indicate a need to loosen the configuration of children’s life stories and identities as ‘pre-care’ and ‘post-care’, and instead move toward an acceptance of children’s multiple selves and multiple important others as existing across time.

Graft‐versus‐host disease (GVHD) is a major complication of allogeneic haematopoietic stem cell transplantation (allo‐HSCT) that develops when donor T cells in the graft become reactive against the host. Post‐transplant cyclophosphamide (PTCy) is increasingly used in mismatched allo‐HSCT, but how PTCy impacts donor T cells and reduces GVHD is unclear. This study aimed to determine the effect of PTCy on reactive human donor T cells and GVHD development in a preclinical humanized mouse model. Immunodeficient NOD‐scid‐IL2Rγ(null) mice were injected intraperitoneally (i.p.) with 20 × 10(6) human peripheral blood mononuclear cells stained with carboxyfluorescein succinimidyl ester (CFSE) (day 0). Mice were subsequently injected (i.p.) with PTCy (33 mg kg(−1)) (PTCy‐mice) or saline (saline‐mice) (days 3 and 4). Mice were assessed for T‐cell depletion on day 6 and monitored for GVHD for up to 10 weeks. Flow cytometric analysis of livers at day 6 revealed lower proportions of reactive (CFSE(low)) human (h) CD3(+) T cells in PTCy‐mice compared with saline‐mice. Over 10 weeks, PTCy‐mice showed reduced weight loss and clinical GVHD, with prolonged survival and reduced histological liver GVHD compared with saline‐mice. PTCy‐mice also demonstrated increased splenic hCD4(+):hCD8(+) T‐cell ratios and reduced splenic Tregs (hCD4(+) hCD25(+) hCD127(lo)) compared with saline‐mice. This study demonstrates that PTCy reduces GVHD in a preclinical humanized mouse model. This corresponded to depletion of reactive human donor T cells, but fewer human Tregs.

Graft-versus-host disease (GVHD) is a major complication that occurs following allogeneic haematopoietic stem cell transplantation (HSCT) for the treatment of haematological cancers and other blood-related disorders. GVHD is an inflammatory disorder, where the transplanted donor immune cells can mediate an immune response against the recipient and attack host tissues. Despite over 60 years of research, broad-range immune suppression is still used to prevent or treat GVHD, leading to an increased risk of cancer relapse and infection. Therefore, further insights into the disease mechanisms and development of predictive and prognostic biomarkers are key to improving outcomes and reducing GVHD development following allogeneic HSCT. An important preclinical tool to examine the pathophysiology of GVHD and to understand the key mechanisms that lead to GVHD development are preclinical humanised mouse models. Such models of GVHD are now well-established and can provide valuable insights into disease development. This review will focus on models where human peripheral blood mononuclear cells are injected into immune-deficient non-obese diabetic (NOD)-scid-interleukin-2(IL-2)Rγ mutant (NOD-scid-IL2Rγnull) mice. Humanised mouse models of GVHD can mimic the clinical setting for GVHD development, with disease progression and tissues impacted like that observed in humans. This review will highlight key findings from preclinical humanised mouse models regarding the role of donor human immune cells, the function of cytokines and cell signalling molecules and their impact on specific target tissues and GVHD development. Further, specific therapeutic strategies tested in these preclinical models reveal key molecular pathways important in reducing the burden of GVHD following allogeneic HSCT.

Background: Physical activity during childhood is associated with multiple short- and long-term health benefits. Physical activity levels decline throughout primary school emphasising a need for effective strategies to promote more activity in children. Children have rarely been involved in the intervention development process. This gap is an important omission and there is much to be learnt from existing qualitative studies with children, which could serve as a starting point for specific projects. This systematic review aimed to synthesise qualitative studies with primary school children in the United Kingdom to identify children’s perspectives on why physical activity is important, the factors that influence their physical activity and what they like when it comes to physical activity. Methods: A search of seven databases (conducted in October 2019) identified 26 papers for inclusion. Data extraction and synthesis were conducted using qualitative thematic synthesis. The quality of papers was assessed using the Critical Appraisal Skills Programme checklist for qualitative research. Results: Across the diverse range of studies, several key themes were identified in relation to the three research questions. Children have a comprehensive understanding of the various benefits of physical activity, including benefits for health, fitness and skills development. A range of social agents and practical issues influence children’s physical activity, with friend and peer influences being particularly salient. Most children like to have choice over the activities they undertake and the opportunities for creative physical play such as making up active games. Conclusions: The findings suggest that future interventions should utilize peer relationships, ensure a variety of activities are offered to cater to a broad range of children’s physical activity preferences and incorporate child-led activities where possible. The included studies also highlight a need for more diversity in qualitative research in this area, particularly in terms of ethnicity and age, and combining traditional qualitative methods with creative methods, such as photography, may provide richer insights than when using a single mode of data collection. We also highlight several methodological challenges, and in particular, the need for greater acknowledgement of the role of the researcher in qualitative work with children.

Background: Advice to families to follow infant care practices known to reduce the risks of Sudden Unexpected Death in Infancy (SUDI) has led to a reduction in deaths across the world. This reduction has slowed in the last decade with most deaths now occurring in families experiencing social and economic deprivation. A systematic review of the literature was commissioned by the National Child Safeguarding Practice Review Panel in England. The review covered three areas: interventions to improve engagement with support services, parental decision-making for the infant sleep environment, and interventions to improve safer sleep practices in families with infants considered to be at risk of SUDI.Aim: To describe the safer sleep interventions tested with families with infants at risk of SUDI and investigate what this literature can tell us about what works to reduce risk and embed safer sleep practices in this group.Methods: Eight online databases were systematically searched in December 2019. Intervention studies that targeted families with infants (0–1 year) at increased risk of SUDI were included. Studies were limited to those from Western Europe, North America or Australasia, published in the last 15 years. The Quality Assessment Tool for Studies with Diverse Designs was applied to assess quality. Data from included studies were extracted for narrative synthesis, including mode of delivery using Michie et al.'s Mode of Delivery Taxonomy.Results: The wider review returned 3,367 papers, with 23 intervention papers. Five types of intervention were identified: (1) infant sleep space and safer sleep education programs, (2) intensive or targeted home visiting services, (3) peer educators/ambassadors, (4) health education/raising awareness interventions, (5) targeted health education messages using digital media.Conclusion: Influencing behavior in families with infants at risk of SUDI has traditionally focused on “getting messages across,” with interventions predominantly using education and awareness raising mechanisms. This review found evidence of interventions moving from “information giving” to “information exchange” models using personalized, longer term relationship-building models. This shift may represent an improvement in how safer sleep advice is implemented in families with infants at risk, but more robust evidence of effectiveness is required.Systematic Review Registration:https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/901091/DfE_Death_in_infancy_review.pdf, identifier: CRD42020165302.

Islet transplantation is an emerging therapy for type 1 diabetes and hypoglycemic unawareness. However, a key challenge for islet transplantation is cellular rejection and the requirement for long-term immunosuppression. In this study, we established a diabetic humanized NOD-scidIL2Rγnull (NSG) mouse model of T-cell–mediated human islet allograft rejection and developed a therapeutic regimen of low-dose recombinant human interleukin-2 (IL-2) combined with low-dose rapamycin to prolong graft survival. NSG mice that had received renal subcapsular human islet allografts and were transfused with 1 × 107 of human spleen mononuclear cells reconstituted human CD45+ cells that were predominantly CD3+ T cells and rejected their grafts with a median survival time of 27 days. IL-2 alone (0.3 × 106 IU/m2 or 1 × 106 IU/m2) or rapamycin alone (0.5–1 mg/kg) for 3 weeks did not prolong survival. However, the combination of rapamycin with IL-2 for 3 weeks significantly prolonged human islet allograft survival. Graft survival was associated with expansion of CD4+CD25+FOXP3+ regulatory T cells (Tregs) and enhanced transforming growth factor-β production by CD4+ T cells. CD8+ T cells showed reduced interferon-γ production and reduced expression of perforin-1. The combination of IL-2 and rapamycin has the potential to inhibit human islet allograft rejection by expanding CD4+FOXP3+ Tregs in vivo and suppressing effector cell function and could be the basis of effective tolerance-based regimens.

The ATP-gated P2X7 ion channel has emerging roles in amyotrophic lateral sclerosis (ALS) progression. Pharmacological blockade of P2X7 with Brilliant Blue G can ameliorate disease in SOD1G93A mice, but recent data suggests that this antagonist displays poor penetration of the central nervous system (CNS). Therefore, the current study aimed to determine whether the CNS-penetrant P2X7 antagonist, JNJ-47965567, could ameliorate ALS progression in SOD1G93A mice. A flow cytometric assay revealed that JNJ-47965567 impaired ATP-induced cation dye uptake in a concentration-dependent manner in murine J774 macrophages. Female and male SOD1G93A mice were injected intraperitoneally with JNJ-47965567 (30 mg/kg) or 2-(hydroxypropyl)-beta-cyclodextrin (vehicle control) three times a week from disease onset until end stage, when tissues were collected and studied. JNJ-47965567 did not impact weight loss, clinical score, motor (rotarod) coordination or survival compared to control mice. NanoString analysis revealed altered spinal cord gene expression in JNJ-47965567 mice compared to control mice, but such differences were not confirmed by quantitative PCR. Flow cytometric analyses revealed no differences between treatments in the frequencies or activation status of T cell or dendritic cell subsets in lymphoid tissues or in the concentrations of serum cytokines. Notably, serum IL-27, IFNβ and IL-10 were present in relatively high concentrations compared to other cytokines in both groups. In conclusion, JNJ-47965567 administered thrice weekly from disease onset did not alter disease progression or molecular and cellular parameters in SOD1G93A mice.

Background Tolerance induced in stringent animal transplant models using donor-specific transfusions (DST) has previously required additional immunological manipulation. Here, we demonstrate a dominant skin-allograft tolerance model induced by a single DST across an major histocompatibility class I mismatch in an unmanipulated B6 host. Methods C57BL/6 (H-2) (B6) mice were injected intravenously with splenocytes from B6.C.H-2 (H-2k) (bm1) or F1 (B6 × bm1) mice before skin transplantation. Mice were transplanted 7 days postinjection with donor (bm1 or F1) and third-party B10.BR (H-2) skin grafts. Results B6 hosts acutely rejected skin grafts from B6.C.H-2 (bm1) and F1 (B6 × bm1) mice. A single transfusion of F1 splenocytes into B6 mice without any additional immune modulation led to permanent acceptance of F1 skin grafts. This graft acceptance was associated with persistence of donor cells long-term in vivo. The more rapid removal of DST bm1 cells than F1 cells was reduced by natural killer-cell depletion. Tolerant grafts survived an in vivo challenge with naive splenocytes. Both CD4CD25 and CD4CD25 T cells from F1 DST treated B6 mice suppressed alloproliferation in vitro. Tolerance was associated with expansion of peripheral Foxp3CD4CD25 regulatory T cells (Treg) and increased forkhead box P3 (Foxp3) expression in tolerant grafts. In tolerant mice, Foxp3 Treg arises from the proliferation of indirectly activated natural Foxp3 Treg (nTreg) and depletion of Foxp3 Treg abrogates skin-graft tolerance. Conclusions This study demonstrates that the persistence of transfused semiallogeneic donor cells mismatched at major histocompatibility class I can enhance tolerance to subsequent skin allografts through indirectly expanded nTreg leading to dominant tolerance without additional immunological manipulation.

Background: Allogeneic haematopoietic stem cell transplantation (HSCT) is a curative therapy for blood cancers; but results in the development of graft-versus-host disease (GVHD) in up to 70% of recipients. During GVHD, tissue damage results in ATP release into the extracellular compartment activating P2X7 on antigen-presenting cells, leading to the release of pro-inflammatory cytokines and subsequent activation of donor T cells. Therefore, the aim of the present study was to examine murine (m) P2rx7 and human (h) P2RX7 gene expression in GVHD target organs of humanised mice, and further characterise disease impact in these organs. Methods: NOD-scid IL2Rγnull (NSG) mice were injected with human peripheral blood mononuclear cells (hu-PBMC-NSG mice) or phosphate-buffered saline (PBS, control). Leucocytes were assessed by flow cytometry; gene expression was measured by quantitative polymerase chain reaction (qPCR), and tissue sections examined by histology. Results: Compared with control mice, hu-PBMC-NSG mice had increased mP2rx7 and mP2rx4 expression in the duodenum, ileum and skin. hP2RX7 was expressed in all tissues examined. hu-PBMC-NSG mice also displayed increased mReg3g expression in the duodenum and ileum, despite limited histological gut GVHD. hu-PBMC-NSG mice showed histological evidence of GVHD in the skin, liver and lung. Compared with control mice, hu-PBMC-NSG mice displayed increased ear swelling. Conclusion: Combined data revealed that P2rx7 is up-regulated in gut and skin GVHD and that P2RX7 is present in target tissues of GVHD, corresponding to human leucocyte infiltration. Data also reveal increased mReg3g expression and ear swelling in hu-PBMC-NSG mice, offering new measurements of early-stage gut GVHD and skin GVHD, respectively.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative method for blood cancers and other blood disorders, but is limited by the development of graft-versus-host disease (GVHD). GVHD results in inflammatory damage to the host liver, gastrointestinal tract and skin, resulting in high rates of morbidity and mortality in HSCT recipients. Activation of the A2A receptor has been previously demonstrated to reduce disease in allogeneic mouse models of GVHD. This study aimed to investigate the effect of A2A activation on disease development in a humanised mouse model of GVHD. Immunodeficient non-obese diabetic-severe combined immunodeficiency-interleukin (IL)-2 receptor γnull (NSG) mice injected with human (h) peripheral blood mononuclear cells (hPBMCs), were treated with either the A2A agonist CGS 21680 or control vehicle. Contrary to the beneficial effect of A2A activation in allogeneic mouse models, CGS 21680 increased weight loss, and failed to reduce the clinical score or increase survival in this humanised mouse model of GVHD. Moreover, CGS 21680 reduced T regulatory cells and increased serum human IL-6 concentrations. Conversely, CGS 21680 reduced serum human tumour necrosis factor (TNF)-α concentrations and leukocyte infiltration into the liver, indicating that A2A activation can, in part, reduce molecular and histological GVHD in this model. Notably, CGS 21680 also prevented healthy weight gain in NSG mice not engrafted with hPBMCs suggesting that this compound may be suppressing appetite or metabolism. Therefore, the potential benefits of A2A activation in reducing GVHD in HSCT recipients may be limited and confounded by adverse impacts on weight, decreased T regulatory cell frequency and increased IL-6 production.

Graft-versus-host disease (GVHD) is a frequent complication following allogeneic hematopoietic stem cell transplantation (HSCT) with current therapies limited to general immunosuppression. Humanized mouse models of GVHD are emerging as valuable intermediaries to allow translation of findings from allogeneic mouse models to humans to prevent and treat this disease, but such models require further characterization. In this study, humanized mice were generated by injecting immunodeficient non-obese diabetic severe combined immunodeficiency interleukin (IL)-2 receptor γ common chain null (NSG) mice with human peripheral blood mononuclear cells (hPBMCs). Clinical GVHD development was assessed using established scoring criteria (weight loss, posture, activity, fur texture and skin integrity). Differences between humanized NSG mice that developed clinical or subclinical GVHD were then compared. Both groups of mice demonstrated similar frequencies of human leukocyte engraftment. In contrast, mice that developed clinical GVHD demonstrated increased histological damage compared to mice with subclinical GVHD. Furthermore, mice with clinical GVHD exhibited increases in the splenic human CD4+:CD8+ T cell ratio, serum human interferon (IFN)-γ and intestinal human IL-17 expression compared to mice with subclinical GVHD. These cellular and molecular changes could be used as potential markers of disease progression in this preclinical model. This study also provides further insights into GVHD development which may be relevant to human HSCT recipients.

CD39 and CD73 are ecto-nucleotidases present on human peripheral blood mononuclear cells (PBMCs) and are emerging biomarkers on these cells in various disorders including cancer. Many factors influence PBMC quality, so it is essential to validate sample processing methods prior to incorporation in clinical studies. This study examined the impact of both PBMC cryopreservation and PBMC isolation using SepMate density gradient centrifugation on CD39 and CD73 expressing subsets. First, PBMCs were isolated from the peripheral blood of 11 healthy donors by routine Ficoll-Paque density gradient centrifugation, cryopreserved and compared with freshly isolated PBMCs by flow cytometry. The proportions of T and B cells expressing combinations of CD39 and CD73 were relatively stable over 6-month cryopreservation, although some T cell combinations revealed small but significant changes. Second, peripheral blood was collected from six healthy donors to compare PBMCs isolated by SepMate or Ficoll-Paque density gradient centrifugation. Compared with Ficoll-Paque, the more rapid SepMate method yielded 9.1% less PBMCs but did not alter cell viability or proportions of T and B cells expressing combinations of CD39 and CD73. The present study reveals that cryopreservation is suitable for studying T and B cells expressing combinations of CD39 and CD73. However, caution should be exercised when observing small differences in these cryopreserved subsets between different cohorts. Further, SepMate and Ficoll-Paque methods of PBMC isolation show similar results for T and B cell subset analysis; however, SepMate is a faster and easier approach.

Regulatory T cells (Tregs) protect against graft-versus-host disease (GVHD), a life-threatening complication of allogeneic hematopoietic stem cell transplantation. The ectoenzyme CD39 is important for increasing the immunosuppressive function of Tregs. The rs10748643 (A → G) single-nucleotide polymorphism (SNP) in intron 1 of the human ENTPD1 gene is associated with increased proportions of CD39+ Tregs. This study aimed to determine whether the rs10748643 SNP corresponded to increased proportions of CD39+ Tregs in an Australian donor population, and whether this SNP influences clinical GVHD in a humanized mouse model. Donors were genotyped for the rs10748643 SNP by Sanger sequencing, and the proportion of CD39+ T cells in donor peripheral blood was determined by flow cytometry. Donors encoding the G allele (donorsAG/GG ) demonstrated higher proportions of CD39+ CD3+ CD4+ CD25+ CD127lo Tregs, but not CD39+ CD3+ CD8+ T cells or CD39+ CD3+ CD4+ conventional T cells, compared with donors homozygous for the A allele (donorsAA ). NOD-SCID-IL2Rγnull mice were injected with human peripheral blood mononuclear cells from either donorsAA (hCD39AA mice) or donorsAG/GG (hCD39AG/GG mice). hCD39AG/GG mice demonstrated significantly greater weight loss and GVHD clinical scores, and significantly reduced survival, compared with hCD39AA mice. hCD39AG/GG mice showed significantly higher hCD4+ :hCD8+ T-cell ratios than hCD39AA mice, but displayed similar proportions of CD3+ hCD4+ hCD25+ hCD127lo Tregs and hCD39+ Tregs. However, the proportion of human Tregs corresponded to survival in hCD39AA mice, but not in hCD39AG/GG mice. This study demonstrates that donors encoding the G allele show higher percentages of CD39+ Tregs, but cause worsened GVHD in humanized mice compared with donors homozygous for the A allele.

Allogeneic haematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for blood cancers and other haematological disorders. However, allo-HSCT leads to graft-versus-host disease (GVHD), a severe and often lethal immunological response, in the majority of transplant recipients. Current therapies for GVHD are limited and often reduce the effectiveness of allo-HSCT. Therefore, pro- and anti-inflammatory factors contributing to disease need to be explored in order to identify new treatment targets. Purinergic signalling plays important roles in haematopoiesis, inflammation and immunity, and recent evidence suggests that it can also affect haematopoietic stem cell transplantation and GVHD development. This review provides a detailed assessment of the emerging roles of purinergic receptors, most notably P2X7, P2Y2 and A2A receptors, and ectoenzymes, CD39 and CD73, in GVHD.

SummaryGraft-versus-host disease (GVHD) remains a major problem after allogeneic haematopoietic stem cell transplantation, a curative therapy for haematological malignancies. Previous studies have demonstrated a role for the adenosine triphosphate (ATP)-gated P2X7 receptor channel in allogeneic mouse models of GVHD. In this study, injection of human peripheral blood mononuclear cells (PBMCs) into immunodeficient non-obese diabetic-severe combined immunodeficiency-interleukin (NOD-SCID-IL)-2Rγnull (NSG) mice established a humanized mouse model of GVHD. This model was used to study the effect of P2X7 blockade in this disease. From five weeks post-PBMC injection, humanized mice exhibited clinical signs and histopathology characteristic of GVHD. The P2X7 antagonist, Brilliant Blue G (BBG), blocked ATP-induced cation uptake into both murine and human cells in vitro. Injection of BBG (50 mg/kg) into NSG mice did not affect engraftment of human leucocytes (predominantly T cells), or the clinical score and survival of mice. In contrast, BBG injection reduced circulating human interferon (IFN)-γ significantly, which was produced by human CD4+ and CD8+ T cells. BBG also reduced human T cell infiltration and apoptosis in target organs of GVHD. In conclusion, the P2X7 antagonist BBG reduced circulating IFN-γ in a humanized mouse model of GVHD supporting a potential role for P2X7 to alter the pathology of this disease in humans.

Psoriasis is a chronic inflammatory skin disorder, characterised by epidermal hyperplasia (acanthosis) and leukocyte infiltration of the skin. Current therapies are inadequate, highlighting the need for new therapeutic targets. The P2X7 receptor is implicated in the pathogenesis of psoriasis. This study investigated the role of P2X7 in imiquimod (IMQ)-induced psoriasis-like inflammation. Topically applied IMQ caused twofold greater ear swelling in BALB/c mice compared to C57BL/6 mice, which encode a partial loss-of-function missense mutation in the P2RX7 gene. However, there was no difference in histological skin pathology (acanthosis and leukocyte infiltration) between the two strains. IMQ treatment up-regulated P2X7 expression in skin from both mouse strains. Additionally, IMQ induced ATP release from cultured human keratinocytes, a process independent of cell death. Injection of the P2X7 antagonist Brilliant Blue G (BBG) but not A-804598 partly reduced ear swelling compared to vehicle-injected control mice. Neither antagonist altered skin pathology. Moreover, no difference in ear swelling or skin pathology was observed between C57BL/6 and P2X7 knock-out (KO) mice. Flow cytometric analysis of IMQ-treated skin from C57BL/6 and P2X7 KO mice demonstrated similar leukocyte infiltration, including neutrophils, macrophages and T cells. In conclusion, this study demonstrates that P2X7 is not essential for development of IMQ-induced psoriasis-like inflammation but does not exclude a role for this receptor in psoriasis development in humans or other mouse models of this disease.

The ATP-gated P2X7 ion channel has emerging roles in amyotrophic lateral sclerosis (ALS) progression. Pharmacological blockade of P2X7 with Brilliant Blue G can ameliorate disease in SOD1(G93A) mice, but recent data suggests that this antagonist displays poor penetration of the central nervous system (CNS). Therefore, the current study aimed to determine whether the CNS-penetrant P2X7 antagonist, JNJ-47965567, could ameliorate ALS progression in SOD1(G93A) mice. A flow cytometric assay revealed that JNJ-47965567 impaired ATP-induced cation dye uptake in a concentration-dependent manner in murine J774 macrophages. Female and male SOD1(G93A) mice were injected intraperitoneally with JNJ-47965567 (30 mg/kg) or 2-(hydroxypropyl)-beta-cyclodextrin (vehicle control) three times a week from disease onset until end stage, when tissues were collected and studied. JNJ-47965567 did not impact weight loss, clinical score, motor (rotarod) coordination or survival compared to control mice. NanoString analysis revealed altered spinal cord gene expression in JNJ-47965567 mice compared to control mice, but such differences were not confirmed by quantitative PCR. Flow cytometric analyses revealed no differences between treatments in the frequencies or activation status of T cell or dendritic cell subsets in lymphoid tissues or in the concentrations of serum cytokines. Notably, serum IL-27, IFNβ and IL-10 were present in relatively high concentrations compared to other cytokines in both groups. In conclusion, JNJ-47965567 administered thrice weekly from disease onset did not alter disease progression or molecular and cellular parameters in SOD1(G93A) mice. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s11302-020-09692-4) contains supplementary material, which is available to authorized users.

Islet transplantation is an emerging therapy for type 1 diabetes and hypoglycemic unawareness. However, a key challenge for islet transplantation is cellular rejection and the requirement for long-term immunosuppression. In this study, we established a diabetic humanized NOD-scidIL2Rγ

Graft-versus-host disease (GVHD) is a life-threatening consequence of allogeneic haematopoietic stem cell transplantation, a curative therapy for haematological malignancies. The ATP-gated P2X7 receptor channel is implicated in the development of GVHD. P2X7 activity on human leukocytes can be influenced by gain-of-function (GOF) and loss-of-function (LOF) single nucleotide polymorphisms (SNPs) in the P2RX7 gene. In this study, the P2RX7 gene was sequenced in 25 human donors and the P2X7 activity on subsets of peripheral blood T cells, natural killer (NK) cells and monocytes was measured using an ATP-induced dye uptake assay. GOF and LOF SNPs representing 10 of the 17 known P2RX7 haplotypes were identified, and correlated with P2X7 activity on all leukocyte subsets investigated. Notably, invariant (i) NK T cells displayed the highest P2X7 activity amongst all cell types studied. To determine if donor P2X7 activity influenced the development of GVHD, immunodeficient NOD-SCID-IL2Rγ(null) (NSG) mice were injected with human peripheral blood mononuclear cells isolated from donors of either GOF (hP2X7(GOF) mice) or LOF (hP2X7(LOF) mice) P2RX7 genotype. Both hP2X7(GOF) and hP2X7(LOF) mice demonstrated similar human leukocyte engraftment, and showed comparable weight loss, GVHD clinical score and overall survival. Donor P2X7 activity did not affect human leukocyte infiltration or GVHD-mediated tissue damage, or the relative expression of human P2X7 or human interferon-γ (hIFNγ) in tissues. Finally, hP2X7(GOF) and hP2X7(LOF) mice demonstrated similar concentrations of serum hIFNγ. This study demonstrates that P2X7 activity correlates with donor P2RX7 genotype on human leukocyte subsets important in GVHD development, but does not affect GVHD development in a humanised mouse model of this disease.

INTRODUCTION Flexible bronchoscopy (FB) is the current gold standard for diagnosing tracheobronchomalacia. However, it is not always feasible and virtual bronchoscopy (VB), acquired from chest multi-detector CT (MDCT) scan is an alternative diagnostic tool. We determined the sensitivity, specificity, and positive and negative predictive values of VB compared to FB in diagnosing tracheobronchomalacia. METHODS Children aged

Allogeneic haematopoietic stem cell transplantation (HSCT) is a frequent curative therapy for numerous haematological malignancies. However, HSCT is limited by the occurrence of graft-versus-host disease (GVHD), with current therapies restricted to general immunosuppression. Activation of the P2X7 receptor by extracellular adenosine triphosphate (ATP) causes inflammation and tissue damage in GVHD. Short-term pharmacological blockade of P2X7 has been shown to reduce clinical disease and/or reduce inflammatory markers in allogeneic and humanized mouse models of GVHD. The current study demonstrates that long-term P2X7 blockade by intra-peritoneal injection of Brilliant Blue G (BBG) thrice weekly for up to 10 weeks did not impact human (h) peripheral blood mononuclear cell (PBMC) engraftment, predominantly T cells, in blood at 3 weeks post-hPBMC injection or in spleens at end-point in humanized mice. Histological analysis demonstrated long-term BBG treatment reduced leukocyte infiltration in the livers of humanized mice. Immunohistochemical analysis demonstrated that BBG treatment reduced liver apoptosis. Long-term BBG treatment did not alter clinical disease, mRNA expression of pro-inflammatory markers in tissues or serum human interferon (IFN)-γ concentrations. Therefore, this study demonstrates that P2X7 activation plays a role in GVHD pathogenesis in the livers of humanized mice, supporting a role for this receptor in GVHD development in HSCT recipients.