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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-IL2Rγ null (NSG) mice were injected intraperitoneally with 2 × 10 7 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., (© 2023 The Authors. Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of the Australian and New Zealand Society for Immunology, Inc.)

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., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2021 John Wiley & Sons Ltd.)

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, P2Y 2 and A 2A receptors, and ectoenzymes, CD39 and CD73, in GVHD.

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., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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 (donors AG/GG ) demonstrated higher proportions of CD39 + CD3 + CD4 + CD25 + CD127 lo Tregs, but not CD39 + CD3 + CD8 + T cells or CD39 + CD3 + CD4 + conventional T cells, compared with donors homozygous for the A allele (donors AA ). NOD-SCID-IL2Rγ null mice were injected with human peripheral blood mononuclear cells from either donors AA (hCD39 AA mice) or donors AG/GG (hCD39 AG/GG mice). hCD39 AG/GG mice demonstrated significantly greater weight loss and GVHD clinical scores, and significantly reduced survival, compared with hCD39 AA mice. hCD39 AG/GG mice showed significantly higher hCD4 + :hCD8 + T-cell ratios than hCD39 AA mice, but displayed similar proportions of CD3 + hCD4 + hCD25 + hCD127 lo Tregs and hCD39 + Tregs. However, the proportion of human Tregs corresponded to survival in hCD39 AA mice, but not in hCD39 AG/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., (© 2020 Australian and New Zealand Society for Immunology Inc.)

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., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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 A 2A receptor has been previously demonstrated to reduce disease in allogeneic mouse models of GVHD. This study aimed to investigate the effect of A 2A activation on disease development in a humanised mouse model of GVHD. Immunodeficient non-obese diabetic-severe combined immunodeficiency-interleukin (IL)-2 receptor γ null (NSG) mice injected with human (h) peripheral blood mononuclear cells (hPBMCs), were treated with either the A 2A agonist CGS 21680 or control vehicle. Contrary to the beneficial effect of A 2A 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 A 2A 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 A 2A 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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Graft-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)-2Rγ null (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., (© 2017 British Society for Immunology.)

Background: Suicidal intent has been described as the seriousness or intensity of the patient's wish to terminate his or her life. Suicide has become an important public health issue throughout the world. It is important to evaluate the intentions of suicide attempts and various psychiatric diagnostic perspectives to understand the multiple dimensions of suicide., Aims: The aim of the work was to study the severity of suicidal intention among suicide attempters in different psychiatric diagnoses and different mode of attempted suicide., Materials and Methods: This study was carried out in the patients, who attempted suicide, by various modes, who were admitted in the wards of KMCTH during 1st January 2007 to 30th December 2007. Suicide Intent Scale (SIS) was used in all the cases that had attempted suicide., Results: Total numbers of patients was 43. Mean SIS was 13.88. The results have shown that majority of cases were female 69.8% (n=30) and male were 30.2% (n=13). The commonest mode of suicide was poisoning 83.7% (n=36) in which moderate suicide intent was 58.3% (n=21); mild suicide intent 33.3% (n=12) and severe suicide intent 8.3% (n=3) (p value < .004). Pesticide (organophosphorus) ingestion was the commonest mode of suicide 44.4% (n=16), followed by pharmacological drugs 33.3% (n=12) (p value < 0.267). The commonest psychiatric diagnosis was depressive disorders 62.9% (n=27), in which moderate suicide intent was found to be maximum 70.4% (n=19) followed by mild suicide intent 14.8% (n=4) and severe suicide intent 14.8% (n=4) (p value < 0.002)., Conclusion: The increasing problem of pesticide poisoning and drug overdose demands strict legal scrutiny in the availability of common means of attempting suicide.

Objective: The aim of the work was to study the socio-demographic variables and their co-morbidity to alcohol consumption and presence of depressive symptomatology., Design: This was a prospective cross-sectional study., Materials and Methods: The study was carried out in 53 patients, who were admitted in the wards of Kathmandu Medical College Teaching Hospital (KMCTH) with the diagnosis of mental and behavioural disorder due to the use of alcohol according to ICD-10. The patients were taken from 1st February 2006 to 30th December 2006. All patients were rated using Hamilton Depression Rating Scale (HDRS). Comparison of alcohol intake, depressive symptomatology and their associations with various socio-demographic variables were done using standard statistical procedures., Results: The present study has shown that more than 94.3% of the patients were suffering from depressive episode. Among all the patients, 11.3% were suffering from severe depressive episode. Alcohol intake was more significantly correlated (p = .002) with Brahmin and Chhetri caste. The other significant correlation of alcohol intake and sociodemographic variable was Nuclear family (p=.001). Among these patients the severity of depression was significantly (p= .001)associated with duration of alcohol intake. Marital status was another important factor affecting comorbidity of alcohol intake and presence of depressive symptoms (p =.002). Students of 10th to 12th grades of school were found to be using alcohol more often (45.3%). Middle socio-economic status (60.4%) was using alcohol more frequently than other socioeconomical classes., Conclusion: Severity of depression and alcohol intake was found to be significantly associated with various socio-demographic variables such as caste, family structure, marital status and educational status.