Post-Transplant Cyclophosphamide Combined with Brilliant Blue G Reduces Graft-versus-Host Disease without Compromising Graft-versus-Leukaemia Immunity in Humanised Mice.

Allogeneic haematopoietic stem cell transplantation (HSCT) leads to the establishment of graft-versus-leukaemia (GVL) immunity, but in many cases also results in the development of graft-versus-host disease (GVHD). This study aimed to determine if P2X7 antagonism using Brilliant Blue G (BBG) could improve the beneficial effects of post-transplant cyclophosphamide (PTCy) in a humanised mouse model of GVHD, without comprising GVL immunity. NOD.Cg-Prkdcscid Il2rgtm1Wjl (NSG) mice were injected with human peripheral blood mononuclear cells (PBMCs) (Day 0), then with cyclophosphamide (33 mg/kg) on Days 3 and 4, and with BBG (50 mg/kg) (or saline) on Days 0-10. PTCy with BBG reduced clinical GVHD development like that of PTCy alone. However, histological analysis revealed that the combined treatment reduced liver GVHD to a greater extent than PTCy alone. Flow cytometric analyses revealed that this reduction in liver GVHD by PTCy with BBG corresponded to an increase in human splenic CD39+ Tregs and a decrease in human serum interferon-γ concentrations. In additional experiments, humanised NSG mice, following combined treatment, were injected with human THP-1 acute myeloid leukaemia cells on Day 14. Flow cytometric analyses of liver CD33+ THP-1 cells showed that PTCy with BBG did not mitigate GVL immunity. In summary, PTCy combined with BBG can reduce GVHD without compromising GVL immunity. Future studies investigating P2X7 antagonism in combination with PTCy may lead to the development of novel treatments that more effectively reduce GVHD in allogeneic HSCT patients without promoting leukaemia relapse.

Tocilizumab increases regulatory T cells, reduces natural killer cells and delays graft-versus-host disease development in humanized mice treated with post-transplant cyclophosphamide.

Graft-versus-host disease (GVHD) is a life-threatening complication following donor hematopoietic stem cell transplantation, where donor T cells damage host tissues. This study investigated the effect of tocilizumab (TOC) combined with post-transplant cyclophosphamide (PTCy) on immune cell engraftment and GVHD development in a humanized mouse model. NOD-scid-IL2Rγnull (NSG) mice were injected intraperitoneally with 2 × 107 human (h) peripheral blood mononuclear cells and cyclophosphamide (33 mg kg-1 ) or saline on days 3 and 4, then TOC or control antibody (0.5 mg mouse-1 ) twice weekly for 28 days. Mice were monitored for clinical signs of GVHD for either 28 or 70 days. Spleens and livers were assessed for human leukocyte subsets, and serum cytokines and tissue histology were analyzed. In the short-term model (day 28), liver and lung damage were reduced in PTCy + TOC compared with control mice. All groups showed similar splenic hCD45+ leukocyte engraftment (55-60%); however, PTCy + TOC mice demonstrated significantly increased (1.5-2-fold) splenic regulatory T cells. Serum human interferon gamma was significantly reduced in PTCy + TOC compared with control mice. Long-term (day 70), prolonged survival was similar in PTCy + TOC (median survival time, > 70 days) and PTCy mice (median survival time, 56 days). GVHD onset was significantly delayed in PTCy + TOC, compared with TOC or control mice. Notably, natural killer cells were reduced (77.5%) in TOC and PTCy + TOC mice. Overall, combining PTCy with TOC increases regulatory T cells and reduces clinical signs of early GVHD, but does not improve long-term survival compared with PTCy alone.

Animal Models for the Investigation of P2X7 Receptors.

The P2X7 receptor is a trimeric ligand-gated cation channel activated by extracellular adenosine 5'-triphosphate. The study of animals has greatly advanced the investigation of P2X7 and helped to establish the numerous physiological and pathophysiological roles of this receptor in human health and disease. Following a short overview of the P2X7 distribution, roles and functional properties, this article discusses how animal models have contributed to the generation of P2X7-specific antibodies and nanobodies (including biologics), recombinant receptors and radioligands to study P2X7 as well as to the pharmacokinetic testing of P2X7 antagonists. This article then outlines how mouse and rat models have been used to study P2X7. These sections include discussions on preclinical disease models, polymorphic P2X7 variants, P2X7 knockout mice (including bone marrow chimeras and conditional knockouts), P2X7 reporter mice, humanized P2X7 mice and P2X7 knockout rats. Finally, this article reviews the limited number of studies involving guinea pigs, rabbits, monkeys (rhesus macaques), dogs, cats, zebrafish, and other fish species (seabream, ayu sweetfish, rainbow trout and Japanese flounder) to study P2X7.

Low Pretreatment CD4+:CD8+ T Cell Ratios and CD39+CD73+CD19+ B Cell Proportions Are Associated with Improved Relapse-Free Survival in Head and Neck Squamous Cell Carcinoma.

The ectonucleotidases CD39 and CD73 are present on immune cells and play important roles in cancer progression by suppressing antitumour immunity. As such, CD39 and CD73 on peripheral blood mononuclear cells (PBMCs) are emerging as potential biomarkers to predict disease outcomes and treatment responses in cancer patients. This study aimed to examine T and B cells, including CD39 and CD73 expressing subsets, by flow cytometry in PBMCs from 28 patients with head and neck squamous cell carcinoma (HNSCC) and to assess the correlation with the treatment modality, human papillomavirus (HPV) status, and relapse-free survival (RFS). The PBMCs were examined pre-, mid-, and post-radiotherapy with concurrent cisplatin chemotherapy or anti-epidermal growth factor receptor antibody (cetuximab) therapy. Combination radiotherapy caused changes to T and B cell populations, including CD39 and CD73 expressing subsets, but no such differences were observed between concurrent chemotherapy and cetuximab. Pretreatment PBMCs from HPV+ patients contained increased proportions of CD39-CD73-CD4+ T cells and reduced proportions of CD39-/+CD73+CD4+ T cells compared to the equivalent cells from HPV- patients. Notably, the pretreatment CD4+:CD8+ T cell ratios and CD39+CD73+CD19+ B cell proportions below the respective cohort medians corresponded with an improved RFS. Collectively, this study supports the notion that CD39 and CD73 may contribute to disease outcomes in HNSCC patients and may assist as biomarkers, either alone or as part of immune signatures, in HNSCC. Further studies of CD39 and CD73 on PBMCs from larger cohorts of HNSCC patients are warranted.

Post-transplant cyclophosphamide limits reactive donor T cells and delays the development of graft-versus-host disease in a humanized mouse model.

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-IL2Rγnull mice were injected intraperitoneally (i.p.) with 20 × 106 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 (CFSElow ) 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+  hCD127lo ) 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.

P2X7 receptor antagonism increases regulatory T cells and reduces clinical and histological graft-versus-host disease in a humanised mouse model.

Graft-versus-host disease (GVHD) is a severe inflammatory response arising from allogeneic haematopoietic stem cell transplantation. Previous studies revealed that antagonism of the P2X7 receptor with Brilliant Blue G (BBG) reduced liver GVHD but did not alter clinical GVHD in a humanised mouse model. Therefore, the present study aimed to trial a modified injection regime using more frequent dosing of BBG to improve outcomes in this model of GVHD. NOD-scid IL2Rγnull (NSG) mice were injected intraperitoneally (i.p.) with 10 × 106 human peripheral blood mononuclear cells (hPBMCs) (day 0), then daily with BBG (50 mg/kg) or saline (days 0-10). BBG significantly reduced clinical score, mortality and histological GVHD compared with saline treatment (endpoint). BBG significantly increased proportions of human regulatory T cells (Tregs) and human B cells and reduced serum human interferon-γ compared with saline treatment prior to development of clinical GVHD (day 21). To confirm the therapeutic benefit of P2X7 antagonism, NSG mice were injected i.p. with 10 × 106 hPBMCs (day 0), then daily with pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (300 mg/kg) or saline (days 0-10). PPADS increased human Treg proportions compared with saline treatment (day 21), but potential clinical benefits were confounded by increased weight loss with this antagonist. To investigate the role of P2X7 antagonism on Treg survival, hPBMCs were cultured in reduced serum conditions to promote cell death. BBG increased proportions of Tregs (and B cells) compared with saline under these conditions. In conclusion, P2X7 antagonism reduces clinical and histological GVHD in a humanised mouse model corresponding to an increase in human Tregs.

Improving engagement with services to prevent Sudden Unexpected Death in Infancy (SUDI) in families with children at risk of significant harm: A systematic review of evidence.

This paper reports part of a wider systematic review commissioned by the English National Safeguarding Panel on Sudden Unexpected Death in Infancy (SUDI). The wider review covered three areas: interventions to improve safer sleep practices in high-risk families, interventions to improve engagement with services and decision making by parents at high risk of SUDI about infant sleep environments. Here, we report the qualitative and quantitative studies reviewed under the engagement strand. Parental engagement is understood to be a multidimensional task for health and social care professionals comprising attitudinal, relational and behavioural components. Following a PROSPERO registered systematic review synthesizing the three strands outlined, 28 papers were found to be relevant in the review of interventions to improve engagement with services in families with children at risk of significant harm through abuse or neglect. No studies were found that specifically focused on engagement of families at high risk for SUDI, so these wider engagement studies were included. The different types of intervention reported in the included studies are described under two broad themes: Enablers (including parental motivation and working with families) and Barriers. Given the focus in the studies on interventions that support parental engagement, the Enablers theme is more extensive than the Barriers reported although all studies noted well-understood barriers. The evidence underpinning these interventions and approaches are reviewed in this paper. We conclude that effective engagement is facilitated by experienced professionals given time to develop supportive non-judgemental relationships with families in their homes, working long-term, linking with communities and other services. While these conclusions have been drawn from wider studies aimed at reducing child maltreatment, we emphasize lessons to be drawn for SUDI prevention work with families with children at risk of significant harm.

Purinergic Signalling in Allogeneic Haematopoietic Stem Cell Transplantation and Graft-versus-Host Disease.

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.

A single-nucleotide polymorphism in the human ENTPD1 gene encoding CD39 is associated with worsened graft-versus-host disease in a humanized mouse model.

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 â†’ 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.

Increased P2X7 expression in the gastrointestinal tract and skin in a humanised mouse model of graft-versus-host disease.

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.

Comparison of peripheral blood mononuclear cell isolation techniques and the impact of cryopreservation on human lymphocytes expressing CD39 and CD73.

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.

The P2X7 receptor antagonist JNJ-47965567 administered thrice weekly from disease onset does not alter progression of amyotrophic lateral sclerosis in SOD1G93A mice.

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, IFNß 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.

Pharmacological blockade of the CD39/CD73 pathway but not adenosine receptors augments disease in a humanized mouse model of graft-versus-host disease.

Allogeneic hematopoietic stem cell transplantation is a curative therapy for a number of hematological malignancies, but is limited by the development of graft-versus-host disease (GVHD). CD39 and CD73 form an ectoenzymatic pathway that hydrolyzes extracellular adenosine 5'-triphosphate (ATP) to adenosine, which respectively exacerbate or alleviate disease in allogeneic mouse models of GVHD. The current study aimed to explore the role of the CD39/CD73 pathway and adenosine receptor (AR) blockade in a humanized mouse model of GVHD. Immunodeficient nonobese diabetic-severe combined immunodeficiency-IL-2 receptor γnull mice were injected with human peripheral blood mononuclear cells, and subsequently injected with the CD39/CD73 antagonist αß-methylene-ADP (APCP) (50 mg kg-1 ) or saline for 7 days, or the AR antagonist caffeine (10 mg kg-1 ) or saline for 14 days. Mice predominantly engrafted human CD4+ and CD8+ T cells, with smaller proportions of human regulatory T cells, invariant natural killer T cells, monocytes and dendritic cells. Neither APCP nor caffeine altered engraftment of these human leukocyte subsets. APCP (CD39/CD73 blockade) augmented GVHD as shown through increased weight loss and worsened liver histology, including increased leukocyte and human T-cell infiltration, and increased apoptosis. This treatment also increased serum human IL-2 concentrations and decreased the frequency of human CD39-  CD73-  CD4+ T cells. In contrast, caffeine (AR blockade) did not alter GVHD severity or human serum cytokine concentrations (IL-2, IL-6, IL-10 or tumor necrosis factor-α). In conclusion, blockade of CD39/CD73 but not ARs augments disease in a humanized mouse model of GVHD. These results indicate that CD39/CD73 blockade maintains sufficient extracellular ATP concentrations to promote GVHD in this model.

The P2X7 receptor is not essential for development of imiquimod-induced psoriasis-like inflammation in mice.

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.

Activation of the P2X7 receptor induces the rapid shedding of CD23 from human and murine B cells.

Activation of the P2X7 receptor by the extracellular damage-associated molecular pattern, adenosine 5'-triphosphate (ATP), induces the shedding of cell surface molecules including the low-affinity IgE receptor, CD23, from human leukocytes. A disintegrin and metalloprotease (ADAM) 10 mediates P2X7-induced shedding of CD23 from multiple myeloma RPMI 8226 B cells; however, whether this process occurs in primary B cells is unknown. The aim of the current study was to determine whether P2X7 activation induces the rapid shedding of CD23 from primary human and murine B cells. Flow cytometric and ELISA measurements showed that ATP treatment of human and murine B cells induced the rapid shedding of CD23. Treatment of cells with the specific P2X7 antagonist, AZ10606120, near-completely impaired ATP-induced CD23 shedding from both human and murine B cells. ATP-induced CD23 shedding was also impaired in B cells from P2X7 knockout mice. The absence of full-length, functional P2X7 in the P2X7 knockout mice was confirmed by immunoblotting of splenic cells, and by flow cytometric measurements of ATP-induced YO-PRO-1(2+) uptake into splenic B and T cells. The broad-spectrum metalloprotease antagonist, BB-94, and the ADAM10 antagonist, GI254023X, impaired P2X7-induced CD23 shedding from both human and murine B cells. These data indicate that P2X7 activation induces the rapid shedding of CD23 from primary human and murine B cells and that this process may be mediated by ADAM10.

Graft-versus-leukaemia immunity is retained following treatment with post-transplant cyclophosphamide alone or combined with tocilizumab in humanised mice.

Objectives: Donor haematopoietic stem cell transplantation treats leukaemia by inducing graft-versus-leukaemia (GVL) immunity. However, this benefit is often mitigated by graft-versus-host disease (GVHD), which is reduced by post-transplant cyclophosphamide (PTCy) alone or combined with tocilizumab (TOC) in humanised mice. This study established a preclinical humanised mouse model of GVL and investigated whether PTCy alone or combined with TOC impacts GVL immunity. Methods: NOD-scid-IL2Rγnull mice were injected with 2 × 107 human peripheral blood mononuclear cells (hPBMCs) on day 0 and with 1 × 106 THP-1 acute myeloid leukaemia cells on day 14. In subsequent experiments, mice were also injected with PTCy (33 mg kg-1) or Dulbecco's phosphate buffered saline (PBS) on days 3 and 4, alone or combined with TOC or control antibody (25 mg kg-1) twice weekly for 28 days. Clinical signs of disease were monitored until day 42. Results: Mice with hPBMCs from three different donors and THP-1 cells showed similar survival, clinical score and weight loss. hCD33+ leukaemia cells were minimal in mice reconstituted with hPBMCs from two donors but present in mice with hPBMCs from a third donor, suggesting donor-specific GVL responses. hPBMC-injected mice treated with PTCy alone or combined with TOC (PTCy + TOC) demonstrated prolonged survival compared to control mice. PTCy alone and PTCy + TOC-treated mice with hPBMCs showed minimal hepatic hCD33+ leukaemia cells, indicating sustained GVL immunity. Further, the combination of PTCy + TOC reduced histological damage in the lung and liver. Conclusion: Collectively, this research demonstrates that PTCy alone or combined with TOC impairs GVHD without compromising GVL immunity.

DNA vaccine encoding CD40 targeted to dendritic cells in situ prevents the development of Heymann nephritis in rats.

The CD40-CD154 costimulatory pathway has been shown to be critical for both T- and B-cell activation in autoimmune disease. Here, we assessed the effects of blocking this pathway using CD40 DNA vaccine enhanced by dendritic cell targeting on the development of active Heymann nephritis, a rat model of human membranous nephropathy. DNA vaccination delivers plasmid DNA encoding the target antigen, either alone or in combination with enhancing elements, to induce both humoral and cellular immune responses. To determine whether CD40 DNA vaccine targeting the encoded CD40 directly to dendritic cells would improve the efficacy of the vaccination against self-protein CD40, we utilized a plasmid encoding a single-chain Fv antibody specific for the dendritic cell-restricted antigen-uptake receptor DEC205 (scDEC), the target gene CD40, and the adjuvant tetanus sequence p30. This vaccine plasmid was compared to a control plasmid without scDEC. Rats vaccinated with scDEC-CD40 had significantly less proteinuria and renal injury than did rats receiving scControl-CD40 and were protected from developing Heymann nephritis. Thus, CD40 DNA vaccination targeted to dendritic cells limits the development of Heymann nephritis.

Change in weight-for-length status during the first three months: relationships to birth weight and implications for metabolic risk.

Weight-for-length during the early postnatal period is a critical predictor of subsequent body composition and metabolic risk. This study was designed to analyze change in weight-for-length status according to birth weight in early infancy. Data were collected for 267 infants enrolled in the Jackson County Women, Infants, and Children (WIC) program. Postnatal measurements were collected at a clinic visit between birth and 12 weeks of age (mean = 5.7 weeks). Changes in WHO z-scores (weight, length, weight-for-length) between birth and the clinic visit were calculated. Infants were classified as exclusively breastfed or as formula-fed. Ethnicity was coded as Hispanic or non-Hispanic. Infants were classified based on birth weight z-score as lower (<-1 SD) or higher (> +1 SD). Multiple regression models tested birth weight, demographic factors, and feeding as predictors of z-score change measures. Demographic factors and feeding were also tested as moderators of the effects of birth weight. Lower birth weight infants displayed an increase in weight-for-length z-score between birth and the clinic visit. Change in weight-for-length was associated with significant increase in weight z-score but not in length z-score. Higher birth weight predicted decrease in weight and length z-scores but did not predict change in weight-for-length. Hispanic ethnicity predicted decrease in length z-score and increase in weight-for-length z-score but did not moderate effects of birth weight. Increase in weight-for-length among lower birth weight infants and persistence of high weight-for-length among higher birth weight infants may reflect phenotypic adjustments that are maladaptive in adverse dietary environments.

CD8+ regulatory T cells induced by T cell vaccination protect against autoimmune nephritis.

Autoreactive T cells play a pivotal role in the pathogenesis of autoimmune kidney disease. T cell vaccination (TCV) may limit autoimmune disease and induce CD8+ regulatory T cells (Tregs). We used Heymann nephritis (HN), a rat model of human membranous nephritis, to study the effects of TCV on autoimmune kidney disease. We harvested CD4+ T cells from renal tubular antigen (Fx1A) -immunized rats and activated these cells in vitro to express the MHC Class Ib molecule Qa-1. Vaccination of Lewis rats with these autoreactive Fx1A-induced T cells protected against HN, whereas control-primed T cells did not. Rats that underwent TCV had lower levels of proteinuria and serum creatinine and significantly less glomerulosclerosis, tubular damage, and interstitial infiltrates. Furthermore, these rats expressed less IFN-γ and IL-6 in splenocytes, whereas the numbers of Tregs and the expression of Foxp3 were unchanged. In vitro cytotoxicity assays showed CD8+ T cell-mediated elimination of Qa-1-expressing CD4+ T cells. In vivo, TCV abrogated the increase in Qa-1-expressing CXCR5+ TFH cells observed in HN compared with controls. Taken together, these results suggest that TCV protects against autoimmune kidney disease by targeting Qa-1-expressing autoreactive CD4+ cells.

Purinergic signalling in graft-versus-host disease.

Allogeneic hematopoietic stem cell transplantation is used to treat blood cancers, but often results in lethal graft-versus-host disease (GVHD). GVHD is an inflammatory disorder mediated by donor leukocytes that damage host tissues. Purinergic signalling plays important roles in GVHD development in mice but studies of these pathways in human GVHD remain limited. P2X7 receptor activation by ATP on host antigen presenting cells contributes to the induction of GVHD, while activation of this receptor on regulatory T cells, myeloid-derived suppressor cells and possibly type 3 innate lymphoid cells results in their loss to promote GVHD progression. In contrast, A2A receptor activation by adenosine on donor T cells serves to restrict GVHD development. These and other purinergic signalling molecules remain potential biomarkers and therapeutic targets in GVHD.