Bone Marrow Cell Transplantation Research Articles

Influence of glycodelin on the formation of immune response at the level of T-helpers and T-regulatory cells in an <i>in vivo</i> experiment

Objective. To study the influence of glycodelin on T-helpers and Tregs level in the process of forming an immune response to the introduction of allogeneic bone marrow (BM) cells in a dynamic experiment on Wistar rats. Materials and methods. The original experimental model "host versus transplant reaction" on male Wistar rats without preliminary conditioning in recipients was used in the study. Animals were administered recombinant glycodelin against the background of allogeneic intraperitoneal transplantation of BM cells in a dynamic experiment. The level of peripheral T-helpers (CD4+) and Tregs (CD4+CD25+FOXP3+), and the expression of FOXP3 in the spleen and mesenteric lymph nodes were assessed. The material was collected on the 3rd and 21st days of the experiment. Results. Glycodelin was shown to reduce the absolute number of T-helpers in the peripheral blood (on the 3rd and 21st days) and to increase the proportion of Tregs on the 21st day of the experiment against the background of the introduction of allogeneic BM cells. It was found out, that glycodelin reduced the level of Tregs in the white pulp of the spleen on the 3rd day of the experiment, while the number of these cells on the 21st day increased, reducing the number of T-helpers at the same time. At the level of the mesenteric lymph nodes, glycodelin reduced the level of T-helpers on the 21st day of the experiment, simultaneously increasing the number of Tregs. In general, a unidirectional and distributed effect of glycodelin on the immune response at the level of T-helpers was observed, that was a decrease of T-helpers, but an increase of Tregs on the 21st day of the experiment. Conclusion. Thus, glycodelin had an immunomodulatory effect on T-helpers and Tregs formation. The vector of the obtained effects was immunosuppressive in nature and contributed to the suppression of the immune response to allogeneic cells.

Sterility of cord blood units: Evaluation of the collection, preparation and conservation process with a view to establishing a bank of stem cells from cord blood at the Biological Resources Center of the Institut Pasteur de Côte d’Ivoire (2024)

Introduction: Cord blood stem cells are used as an alternative to bone marrow and peripheral blood cell transplantation. For this purpose, cord blood banks have been established worldwide for the collection and cryopreservation of cord blood units. These banks must implement rigorous protocols to ensure the microbiological safety of grafts. The aim of this study is to evaluate the process of selecting mothers and collecting and processing these samples developed for this purpose. Methodology: Pregnant women were enrolled in the study on the basis of selection criteria. Maternal blood and cord blood samples were collected by the in utero method. Serological analyses of maternal blood for the detection of vertically transmitted infections and microbiological examinations of cord blood units were carried out. Results: 31 women out of 46 (68.4%) were enrolled. All had negative serology for the targeted pathogens. Similarly, microbiology revealed no microbial contamination of the cord blood units. Conclusion: This work demonstrates that the procedures for selecting mothers and processing implementations are effective in ensuring the microbiological safety of cord blood units and could be suggested in the context of banking cord blood stem cells at the Institut Pasteur de Côte d’Ivoire.

IL-33/ST2 signaling in monocyte-derived macrophages maintains blood-brain barrier integrity and restricts infarctions early after ischemic stroke

BackgroundBrain microglia and infiltrating monocyte-derived macrophages are vital in preserving blood vessel integrity after stroke. Understanding mechanisms that induce immune cells to adopt vascular-protective phenotypes may hasten the development of stroke treatments. IL-33 is a potent chemokine released from damaged cells, such as CNS glia after stroke. The activation of IL-33/ST2 signaling has been shown to promote neuronal viability and white matter integrity after ischemic stroke. The impact of IL-33/ST2 on blood-brain barrier (BBB) integrity, however, remains unknown. The current study fills this gap and reveals a critical role of IL-33/ST2 signaling in macrophage-mediated BBB protection after stroke.MethodsTransient middle cerebral artery occlusion (tMCAO) was performed to induce ischemic stroke in wildtype (WT) versus ST2 knockout (KO) male mice. IL-33 was applied intranasally to tMCAO mice with or without dietary PLX5622 to deplete microglia/macrophages. ST2 KO versus WT bone marrow or macrophage cell transplantations were used to test the involvement of ST2+ macrophages in BBB integrity. Macrophages were cocultured in transwells with brain endothelial cells (ECs) after oxygen-glucose deprivation (OGD) to test potential direct effects of IL33-treated macrophages on the BBB in vitro.ResultsThe ST2 receptor was expressed in brain ECs, microglia, and infiltrating macrophages. Global KO of ST2 led to more IgG extravasation and loss of ZO-1 in cerebral microvessels 3 days post-tMCAO. Intranasal IL-33 administration reduced BBB leakage and infarct severity in microglia/macrophage competent mice, but not in microglia/macrophage depleted mice. Worse BBB injury was observed after tMCAO in chimeric WT mice reconstituted with ST2 KO bone marrow, and in WT mice whose monocytes were replaced by ST2 KO monocytes. Macrophages treated with IL-33 reduced in vitro barrier leakage and maintained tight junction integrity after OGD. In contrast, IL-33 exerted minimal direct effects on the endothelial barrier in the absence of macrophages. IL-33-treated macrophages demonstrated transcriptional upregulation of an array of protective factors, suggesting a shift towards favorable phenotypes.ConclusionOur results demonstrate that early-stage IL-33/ST2 signaling in infiltrating macrophages reduces the extent of acute BBB disruption after stroke. Intranasal IL-33 administration may represent a new strategy to reduce BBB leakage and infarct severity.

Deletion of TP signaling in macrophages delays liver repair following APAP-induced liver injury by reducing accumulation of reparative macrophage and production of HGF

BackgroundAcetaminophen (APAP)-induced liver injury is the most common cause of acute liver failure. Macrophages are key players in liver restoration following APAP-induced liver injury. Thromboxane A2 (TXA2) and its receptor, thromboxane prostanoid (TP) receptor, have been shown to be involved in tissue repair. However, whether TP signaling plays a role in liver repair after APAP hepatotoxicity by affecting macrophage function remains unclear.MethodsMale TP knockout (TP−/−) and C57BL/6 wild-type (WT) mice were treated with APAP (300 mg/kg). In addition, macrophage-specific TP-knockout (TP△mac) and control WT mice were treated with APAP. We explored changes in liver inflammation, liver repair, and macrophage accumulation in mice treated with APAP.ResultsCompared with WT mice, TP−/− mice showed aggravated liver injury as indicated by increased levels of alanine transaminase (ALT) and necrotic area as well as delayed liver repair as indicated by decreased expression of proliferating cell nuclear antigen (PCNA). Macrophage deletion exacerbated APAP-induced liver injury and impaired liver repair. Transplantation of TP-deficient bone marrow (BM) cells to WT or TP−/− mice aggravated APAP hepatotoxicity with suppressed accumulation of macrophages, while transplantation of WT-BM cells to WT or TP−/− mice attenuated APAP-induced liver injury with accumulation of macrophages in the injured regions. Macrophage-specific TP−/− mice exacerbated liver injury and delayed liver repair, which was associated with increased pro-inflammatory macrophages and decreased reparative macrophages and hepatocyte growth factor (HGF) expression. In vitro, TP signaling facilitated macrophage polarization to a reparative phenotype. Transfer of cultured BM-derived macrophages from control mice to macrophage-specific TP−/− mice attenuated APAP-induced liver injury and promoted liver repair. HGF treatment mitigated APAP-induced inflammation and promoted liver repair after APAP-induced liver injury.ConclusionsDeletion of TP signaling in macrophages delays liver repair following APAP-induced liver injury, which is associated with reduced accumulation of reparative macrophages and the hepatotrophic factor HGF. Specific activation of TP signaling in macrophages may be a potential therapeutic target for liver repair and regeneration after APAP hepatotoxicity.

Blockade of PD-1 and TIM-3 Ameliorates CD8+ T Cell Exhaustion in a Mouse Model of Chronic Myeloid Leukemia.

The immune system plays a pivotal role in controlling chronic myeloid leukemia (CML). CD8+ T cell exhaustion results in reduced effectiveness of T cell-mediated immunity, thereby contributing to disease progression. This study intends to figure out whether the combined blockade of inhibitory molecules TIM-3/PD-1 can affect CD8+ T cell exhaustion in CML. A CML mouse model was established via transplantation of bone marrow cells transduced with BCR-ABL-expressing retrovirus vectors. PD-1 and TIM-3 signaling were blocked using corresponding molecular antibodies. Flow cytometry analysis was conducted to detect cell surface molecules and intracellular cytokines. ELISA was employed for measuring cytokine concentrations in the culture medium. The results showed that TIM-3 and PD-1 were coexpressed on exhausted CD8+ T cells from CML mice. Combined blockade of PD-1/TIM3 synergistically delayed CML progression in mice. Moreover, ex vivo experiments showed that their co-blockade promoted the proliferation and cytokine secretion of CD8+ T cells isolated from CML mice. In conclusion, blocking TIM-3 and PD-1 improves exhausted CD8+ T cell function in CML.

Direct Water-Soluble Molecules Transfer from Transplanted Bone Marrow Mononuclear Cell to Hippocampal Neural Stem Cells.

Intravascularly transplanted bone marrow cells, including bone marrow mononuclear cells (BM-MNC) and mesenchymal stem cells, transfer water-soluble molecules to cerebral endothelial cells via gap junctions. After transplantation of BM-MNC, this fosters hippocampal neurogenesis and enhancement of neuronal function. Herein, we report the impact of transplanted BM-MNC on neural stem cells (NSC) in the brain. Surprisingly, direct transfer of water-soluble molecules from transplanted BM-MNC and peripheral mononuclear cells to NSC in the hippocampus was observed already 10 min after cell transplantation, and transfer from BM-MNC to GFAP-positive cortical astrocytes was also observed. In vitro investigations revealed that BM-MNC abolish the expression of hypoxia-inducible factor-1α in astrocytes. We suggest that the transient and direct transfer of water-soluble molecules between cells in circulation and NSC in the brain may be one of the biological mechanisms underlying the repair of brain function.

Comparative effectiveness of mesenchymal stem cell versus bone-marrow mononuclear cell transplantation in heart failure: a meta-analysis of randomized controlled trials

BackgroundThere is no clear evidence on the comparative effectiveness of bone-marrow mononuclear cell (BMMNC) vs. mesenchymal stromal cell (MSC) stem cell therapy in patients with chronic heart failure (HF).MethodsUsing a systematic approach, eligible randomized controlled trials (RCTs) of stem cell therapy (BMMNCs or MSCs) in patients with HF were retrieved to perform a meta-analysis on clinical outcomes (major adverse cardiovascular events (MACE), hospitalization for HF, and mortality) and echocardiographic indices (including left ventricular ejection fraction (LVEF)) were performed using the random-effects model. A risk ratio (RR) or mean difference (MD) with corresponding 95% confidence interval (CI) were pooled based on the type of the outcome and subgroup analysis was performed to evaluate the potential differences between the types of cells.ResultsThe analysis included a total of 36 RCTs (1549 HF patients receiving stem cells and 1252 patients in the control group). Transplantation of both types of cells in patients with HF resulted in a significant improvement in LVEF (BMMNCs: MD (95% CI) = 3.05 (1.11; 4.99) and MSCs: MD (95% CI) = 2.82 (1.19; 4.45), between-subgroup p = 0.86). Stem cell therapy did not lead to a significant change in the risk of MACE (MD (95% CI) = 0.83 (0.67; 1.06), BMMNCs: RR (95% CI) = 0.59 (0.31; 1.13) and MSCs: RR (95% CI) = 0.91 (0.70; 1.19), between-subgroup p = 0.12). There was a marginally decreased risk of all-cause death (MD (95% CI) = 0.82 (0.68; 0.99)) and rehospitalization (MD (95% CI) = 0.77 (0.61; 0.98)) with no difference among the cell types (p > 0.05).ConclusionBoth types of stem cells are effective in improving LVEF in patients with heart failure without any noticeable difference between the cells. Transplantation of the stem cells could not decrease the risk of major adverse cardiovascular events compared with controls. Future trials should primarily focus on the impact of stem cell transplantation on clinical outcomes of HF patients to verify or refute the findings of this study.

Restoration of thymic T-cell development by bone marrow transplantation in mouse radiation lymphomagenesis.

Fractionated total body irradiation (TBI) with X-rays induces thymic lymphoma/leukemia (TL) in C57BL/6 mice. Radiation-induced mouse TL (RITL) can be prevented by bone marrow transplantation (BMT) of unirradiated BM cells. However, the mechanisms underlying the prevention of RITL with BMT remain unclear. Here, we show that BMT restores thymic T-cell differentiation in mice subjected to TBI. TBI (four times of 1.8Gy X-rays weekly) was conducted with C57BL/6 mice. BMT was performed immediately after the last irradiation of TBI in mice by transplantation of BM cells isolated from enhanced green fluorescence protein (eGFP) transgenic mice. Thymic cell numbers were drastically decreased in TBI and TBI + BMT mice compared to those in non-irradiated mice. Flow cytometry showed a dramatic decrease in double negative (DN, CD4-CD8-) thymocytes, especially DN2 (CD25+CD44+) and DN3 (CD25+CD44-) subpopulations, in the TBI mice on Day 10 after the last irradiation. In contrast, the DN2 and DN3 populations were recovered in TBI + BMT mice. Interestingly, these restored DN2 and DN3 cells mainly differentiated from eGFP-negative recipient cells but not from eGFP-positive donor cells, suggesting that transplanted BM cells may interact with recipient cells to restore thymic T-cell development in the RITL model. Taken together, our findings highlight the significance of restoring thymic T-cell differentiation by BMT in RITL prevention.

The impact of infused red blood cell volume on major and bidirectional ABO-mismatched bone marrow transplantation

Background aimsABO incompatibility does not hinder bone marrow transplantation (BMT), but it has been associated with worse outcomes and additional adverse events. This study aimed to verify the impact of incompatible red blood cells (iRBCs) in allogeneic BMT and to determine a safe number of iRBCs to be infused. MethodsWe compared ABO-incompatible (iABO) allogeneic BMT (n = 42) with ABO-compatible allogeneic BMT (n = 44) and evaluated the impact of the number of infused iRBCs on outcomes and adverse events. ResultsThe iABO patients demonstrated delayed time to transfusion independence at 30 days and 60 days, increased requirement for red blood cell (RBC) transfusion and greater hemolysis signals and incidence of pure red cell aplasia. Neutrophil/platelet engraftment, length of hospitalization post-transplant, platelet units required, graft-versus-host disease occurrence and overall survival were similar in both groups. Patients in the iABO group received 1.03 × 1010 iRBCs/kg (range, 0.36–3.88). Infusion of iRBCs >1.0 × 1010 /kg was related to graft failure or death before neutrophil engraftment or platelet engraftment or both as well as increased plasma requirement and increased creatinine. Our results also suggest that antibody titers impact the transplantation scenario. ConclusionsThe iABO transplantation showed some unfavorable outcomes. It is important to monitor the value of iRBCs to be infused, considering the recipient antibody titers. We propose using the number of iRBCs (iRBCs/kg) as a dose parameter with regard to infused iRBCs. Further studies are necessary to clarify the maximum safe number of iRBCs in iABO transplants.

Atorvastatin Treatment Significantly Increased the Concentration of Bone Marrow-Derived Mononuclear Cells and Transcutaneous Oxygen Pressure and Lowered the Pain Scale after Bone Marrow Cells Treatment in Patients with "No-Option" Critical Limb Ischaemia.

The present study investigated the outcomes and possible predictive factors of autologous bone marrow cells (BMCs) therapy in patients with "no-option" critical limb ischaemia (CLI). It was focused on exploring the clinical background and prior statin and renin-angiotensin system (RAS)-acting agents pharmacotherapy related to the therapeutic efficacy of BMCs treatment. In the present study, we reviewed thirty-three patients (mean age 64.9 ± 10 years; 31 males) with advanced CLI after failed or impossible revascularisation, who were treated with 40 mL of autologous BMCs by local intramuscular application. Patients with limb salvage and wound healing (N = 22) were considered as responders to BMCs therapy, and patients with limb salvage and complete ischemic wound healing (N = 13) were defined as super-responders. Logistic regression models were used to screen and identify the prognostic factors, and a receiver operating characteristics (ROC) curve, a linear regression, and a survival curve were drawn to determine the predictive accuracy, the correlation between the candidate predictors, and the risk of major amputation. Based on the univariate regression analysis, baseline C-reactive protein (CRP) and transcutaneous oxygen pressure (TcPO2) values were identified as prognostic factors of the responders, while CRP value, ankle-brachial index (ABI), and bone marrow-derived mononuclear cells (BM-MNCs) concentration were identified as prognostic factors of the super-responders. An area under the ROC curve of 0.768 indicated good discrimination for CRP > 8.1 mg/L before transplantation as a predictive factor for negative clinical response. Linear regression analysis revealed a significant dependence between the levels of baseline CRP and the concentration of BM-MNCs in transplanted bone marrow. Patients taking atorvastatin before BMCs treatment (N = 22) had significantly improved TcPO2 and reduced pain scale after BMCs transplant, compared to the non-atorvastatin group. Statin treatment was associated with reduced risk for major amputation. However, the difference was not statistically significant. Statin use was also associated with a significantly higher concentration of BM-MNCs in the transplanted bone marrow compared to patients without statin treatment. Patients treated with RAS-acting agents (N = 20) had significantly reduced pain scale after BMCs transplant, compared to the non-RAS-acting agents group. Similar results, reduced pain scale and improved TcPO2, were achieved in patients treated with atorvastatin and RAS-acting agents (N = 17) before BMCs treatment. Results of the Spearman correlation showed a significant positive correlation between CLI regression, responders, and previous therapy before BMCs transplant with RAS-acting agents alone or with atorvastatin. CRP and TcPO2 were prognostic factors of the responders, while CRP value, ABI, and BM-MNCs concentration were identified as predictive factors of the super-responders. Atorvastatin treatment was associated with a significantly increased concentration of BM-MNCs in bone marrow concentrate and higher TcPO2 and lower pain scale after BMCs treatment in CLI patients. Similarly, reduced pain scales and improved TcPO2 were achieved in patients treated with atorvastatin and RAS-acting agents before BMCs treatment. Positive correlations between responders and previous treatment before BMCs transplant with RAS-acting agents alone or with atorvastatin were significant.

Allogeneic transplantation of bone marrow versus peripheral blood stem cells from HLA-identical sibling donors for hematological malignancies in 6064 adults from 2003 to 2020: different impacts on survival according to time period

BackgroundMobilized peripheral blood stem cells (PBSC) have been widely used instead of bone marrow (BM) as the graft source for allogeneic hematopoietic cell transplantation (HCT). Although early studies demonstrated no significant differences in survival between PBSC transplantation (PBSCT) and BM transplantation (BMT) from human leukocyte antigen (HLA)-identical sibling donors to adults with hematological malignancies, recent results have been unclear. ObjectiveThe objective of this retrospective study was to compare overall survival (OS), relapse, non-relapse mortality (NRM), hematopoietic recovery and graft-versus-host disease (GVHD) between PBSCT and BMT according to the time period of HCT (2003–2008, 2009–2014, or 2015–2020). Study designWe retrospectively compared the outcomes after PBSCT versus BMT in 6064 adults with hematological malignancies using a Japanese registry database between 2003 and 2020. ResultsThe adjusted probability of OS was significantly higher in BMT recipients compared to PBSCT recipients during the early period of 2003–2008 (adjusted hazard ratio [HR], 0.79; 95% confidence interval [CI], 0.70–0.91; P < 0.001) and the middle period of 2009–2014 (adjusted HR, 0.80; 95% CI, 0.70–0.91; P < 0.001). However, during the late period of 2015–2020, the adjusted probability of OS was comparable between BMT and PBSCT recipients (adjusted HR, 0.94; 95% CI, 0.79–1.13; P = 0.564), which were mainly due to the reduction of NRM. There was no significant difference in the relapse rate between the groups, irrespective of the time period. Compared to BMT, PBSCT led to faster neutrophil and platelet recovery and the cumulative incidences of grades II–IV and grades III–IV acute and overall and extensive chronic GVHD were significantly higher in PBSCT recipients, irrespective of the time period. ConclusionsPBSCT and BMT had similar survival outcomes and relapse rates in adult patients with hematological malignancies during the late time period of 2015–2020 despite the hematopoietic recovery and acute and chronic GVHD being higher in PBSCT recipients in all time periods.

A study of the relationship of the dynamics of development and characteristics of chimerism with manifestations of graft-vs.-host disease in the organs of mice after allogeneic transplantation of whole bone marrow

In the сlinical practice, allogeneiс bone marrow transplantation (BMT) is often cause of the graft-versus-host disease (GvHD). GvHD is explained by the fact that T-lymphocytes, which are administered simultaneously with hematopoietic cells during transplantation and after then formed and matured in the timus of the recipient from donor progenitor cells, recognize and attack the cells of the host. However, a complete explanation of the phenomenon of the GvHD does not exists, and the chimerization of the recipient’s organism as a possible cause of damage of its organs is not taken into account. Therefore, the aim of this work was the modeling of allogeneic transplantation of the whole bone marrow (BM, experiment) and comparing its results with syngeneic transplantation (control) basing on the investigation of engraftment of cells of donor origin in the main GvHD target organs. Bone marrow (BM) donors were Tg(ACTB-EGFP)1Osb/J mice carrying a green fluorescent protein gene (EGFP), recipients were the animal of CBA and C57BL/6 inbred strains with age 2–10 months. 1 day before BMT (1.5×107 cells per mouse) all recipients were irradiated at a dose of 6.5 Gy (LD 50/30). After 1, 3, 5, 7, 11, 14, 21, 28, 35 and 55 days the development of chimerism in the liver, skin and colon of animals was examined using a fluorescent microscope. Already in 1 day, single fibroblast-like donor cells were found in the colon, in 7 days – in the skin and liver. 14–28 days after BMT, with donor cells mainly stroma in the liver, in the skin fibroblasts and keratinocytes were formed, in the colon villous cells and also stromal and parenchymal cells of Peyer’s patches which were died off after irradiation were substituted. Unlike control, in the experimental groups GFP+ giant fibroblasts about 30 mkm in length were found in the stroma of the liver, in the skin and in the colon; in the liver there was a lot of GFP+-bulkheads and fibroblast-like Ito’s cells of a very intricate configuration. To 35–55 days after allogeneic BMT cells of the donor origin in the liver and in the villi of the colon began to destroy, the villi became overgrown with GFP+-connective tissue cells and warped, wall of the colon became thin and the skin was fully substituted with a new one (all these things were never observed in the conrol groups). We propose a hypothesis that beside with GvHD traits like thinning of the colon wall and plenty of roundish GFP+-cells on inner surface of the skin, other signs of the studied after allogeneic BMT organs suggest that the cells of the organs which are formed from mesenchymal stem cells of the whole bone marrow become target for the recipient’s T-cells, i.e. suggest existence of host-versus-graft (HVG) reaction. Obvious manifestation of immune reactions after BMT directly coincides with the term of massive engraftment of the studied organs with cells of donor origin and restoration of the host’s own immune system, i.e. the development of chimerism determines the development of organ damage. This explains the events of GvHD from medical practice – atrophy of the mucous membranes, excess production of collagen, sclerosis of the bile ducts, skin damage, colitis – and the timing of its manifestation.

Risk factors for mortality in children with hemorrhagic cystitis after hematopoietic transplant

Hemorrhagic cystitis (HC) is a devastating complication of bone marrow (BMT) and stem cell transplant (SCT). Much of the literature has focused on exclusively adult patient populations, with limited evidence regarding risk factors for mortality and morbidity among pediatric HC patients. To examine factors associated with all-cause mortality in children with HC after BMT/SCT. The Pediatric Health Information System database was queried for patients with ICD-9/10 codes for hematopoietic transplant and gross hematuria, hematuria unspecified, or cystitis with hematuria. Multivariable logistic regression examined association of medical and surgical interventions frequently employed for hemorrhagic cystitis with mortality and genitourinary morbidity, defined as having received instillation of any bladder medication or having undergone any genitourinary procedure. A total of 811 patients, mean age of 12.4 years and 62% male, were included. Primary diagnosis included 388 (49%) leukemia/lymphoma, 182 (22%) blood dyscrasia, 99 (12%) solid organ tumor, 27 (3%) metabolic disease, 115 (14%) unknown. Transplant type included 377 (46%) bone marrow, 329 (41%) stem cell, 105, and (13%) unknown. Performing any bladder instillation (p<0.0001) or any type of GU procedure (p<0.0001) was significantly associated with mortality. On multivariate analysis, dialysis (OR=10.7, 95% CI=5.7-20.2), genitourinary morbidity (OR=4, 95% CI=2.2-6.8) and intravenous cidofovir (OR=2.0, 95% CI=1.2-3.3) were significantly associated with all cause mortality. Having an underlying diagnosis of blood dyscrasia was protective against mortality (OR=0.425, CI=0.205-0.88). In this large retrospective study evaluating factors associated with mortality in children with HC, all cause mortality was found to be 11%. This is probably an underrepresentation of true mortality in this population, as many patients discharged from the hospital likely die outside the hospital at home or hospice care. This study supports the current literature that invasive GU procedures are not associated with increased survival in patients with severe HC. This study is limited by retrospective use of a billing database that has the potential for errors in data entry and missing data. Patients who were discharged from the hospital were not captured by the PHIS which only collects data from inpatient stays. Patients with HC who received dialysis, intravenous cidofovir, or underwent GU intervention had significantly higher all-cause mortality. High grade HC is a marker of disease severity and efforts should be made by urologists and oncologists to maximize quality of life and limit futile treatments in this patient population.

Aromatase deficiency in transplanted bone marrow cells improves vertebral trabecular bone quantity, connectivity, and mineralization and decreases cortical porosity in murine bone marrow transplant recipients.

Estradiol is an important regulator of bone accumulation and maintenance. Circulating estrogens are primarily produced by the gonads. Aromatase, the enzyme responsible for the conversion of androgens to estrogen, is expressed by bone marrow cells (BMCs) of both hematopoietic and nonhematopoietic origin. While the significance of gonad-derived estradiol to bone health has been investigated, there is limited understanding regarding the relative contribution of BMC derived estrogens to bone metabolism. To elucidate the role of BMC derived estrogens in male bone, irradiated wild-type C57BL/6J mice received bone marrow cells transplanted from either WT (WT(WT)) or aromatase-deficient (WT(ArKO)) mice. MicroCT was acquired on lumbar vertebra to assess bone quantity and quality. WT(ArKO) animals had greater trabecular bone volume (BV/TV p = 0.002), with a higher trabecular number (p = 0.008), connectivity density (p = 0.017), and bone mineral content (p = 0.004). In cortical bone, WT(ArKO) animals exhibited smaller cortical pores and lower cortical porosity (p = 0.02). Static histomorphometry revealed fewer osteoclasts per bone surface (Oc.S/BS%), osteoclasts on the erosion surface (ES(Oc+)/BS, p = 0.04) and low number of osteoclasts per bone perimeter (N.Oc/B.Pm, p = 0.01) in WT(ArKO). Osteoblast-associated parameters in WT(ArKO) were lower but not statistically different from WT(WT). Dynamic histomorphometry suggested similar bone formation indices' patterns with lower mean values in mineral apposition rate, label separation, and BFR/BS in WT(ArKO) animals. Ex vivo bone cell differentiation assays demonstrated relative decreased osteoblast differentiation and ability to form mineralized nodules. This study demonstrates a role of local 17β-estradiol production by BMCs for regulating the quantity and quality of bone in male mice. Underlying in vivo cellular and molecular mechanisms require further study.

Effect of peptides of trophoblastic Β1 glycoprotein on peripheral and local T-regulatory lymphocytes level in Wistar rats with allogeneous bone marrow cell transplantation

Objective. The range of peptide drugs is expanding, but no drug with immunosuppressive activity has yet been found. Considering the fact that the trophoblastic β1-glycoprotein (PSG) is a fetoplacental protein with immunosuppressive activity, short peptide fragments of this protein were studied in the formation of an immune response in a situation of allogeneic cell transplantation. To study the effect of PSG peptides (YECE, YQCE, YVCS, and YACS) on the levels of peripheral and local T-regulatory cells (Treg) during the formation of an immune response to the introduction of allogeneic bone marrow cells (BM) in a dynamic experiment on Wistar rats.&#x0D; Materials and methods. We used an original host-versus-graft model in male Wistar rats without preconditioning of recipients. Animals were injected with PSG peptide fragment composition against a background of allogeneic intraperitoneal transplantation of BM cells in a dynamic experiment, in which the following parameters were evaluated: the level of peripheral "true" Tregs (CD4+CD25+FOXP3+), CD4+CD25-FOXP3+ cells, and FOXP3 expression in mesenteric lymph nodes. Material was collected on days 3 and 21 of the experiment.&#x0D; Results. PSG peptide administration against a background of allogeneic BM cells was found to reduce the absolute and relative amount of Treg in the peripheral blood of rats on days 3 and 21 of the experiment. PSG peptides against the background of the introduction of allogeneic BM cells reduced the absolute and relative amounts of CD4+CD25-FOXP3+ cells on the day 3 of the experiment. The introduction of PSG peptides against the background of the introduction of BM cells resulted in a relative decrease in FOXP3 expression in the T zone of mesenteric lymph nodes on the day 21 of the experiment.&#x0D; Conclusions. Thus, the PSG peptides did not have the expected effect on the level of peripheral and local Treg cells; moreover, the presence of the peptides led to a decrease in the number of these cells.

Muscularis macrophages controlled by NLRP3 maintain the homeostasis of excitatory neurons.

Peristaltic movements in gut are essential to propel ingested materials through the gastrointestinal tract. Intestinal resident macrophages play an important role in this physiological function through protecting enteric neurons. However, it is incompletely clear how individuals maintain the homeostasis of gut motility. Here we found that NLRP3 is a critical factor in controlling loss of muscularis resident macrophages (MMs), and demonstrate that MMs are involved in the homeostasis of excitatory neurons such as choline acetyltransferase (ChAT)+ and vesicular glutamate transporter 2 (VGLUT2)+ but not inhibitory neuronal nitric oxide synthase (nNOS)+ neurons. NLRP3 knockout (KO) mice had enhanced gut motility and increased neurons, especially excitatory ChAT+ and VGLUT2+ neurons. Single cell analyses showed that there had increased resident macrophages, especially MMs in NLRP3 KO mice. The MM proportion in the resident macrophages was markedly higher than those in wild-type (WT) or caspase 1/11 KO mice. Deletion of the MMs and transplantation of the NLRP3 KO bone marrow cells showed that survival of the gut excitatory ChAT+ and VGLUT2+ neurons was dependent on the MMs. Gut microbiota metabolites β-hydroxybutyrate (BHB) could promote gut motility through protecting MMs from pyroptosis. Thus, our data suggest that MMs regulated by NLRP3 maintain the homeostasis of excitatory neurons.

The bone marrow of mouse-rat chimeras contains progenitors of multiple pulmonary cell lineages.

Radiation-induced lung injury (RILI) is a common complication of anti-cancer treatments for thoracic and hematologic malignancies. Bone marrow (BM) transplantation restores hematopoietic cell lineages in cancer patients. However, it is ineffective in improving lung repair after RILI due to the paucity of respiratory progenitors in BM transplants. In the present study, we used blastocyst injection to create mouse-rat chimeras, these are artificial animals in which BM is enriched with mouse-derived progenitor cells. FACS-sorted mouse BM cells from mouse-rat chimeras were transplanted into lethally irradiated syngeneic mice, and the contribution of donor cells to the lung tissue was examined using immunostaining and flow cytometry. Donor BM cells provided long-term contributions to all lung-resident hematopoietic cells which includes alveolar macrophages and dendritic cells. Surprisingly, donor BM cells also contributed up to 8% in pulmonary endothelial cells and stromal cells after RILI. To identify respiratory progenitors in donor BM, we performed single-cell RNA sequencing (scRNAseq). Compared to normal mouse BM, increased numbers of hematopoietic progenitors were found in the BM of mouse-rat chimeras. We also identified unique populations of hemangioblast-like progenitor cells expressing Hes1, Dntt and Ebf1, along with mesenchymal stromal cells expressing Cpox, Blvrb and Ermap that were absent or ultra-rare in the normal mouse BM. In summary, by using rats as "bioreactors", we created a unique mouse BM cell transplant that contributes to multiple respiratory cell types after RILI. Interspecies chimeras have promise for future generations of BM transplants enriched in respiratory progenitor cells.

Impact of stem cell selection between bone marrow and peripheral blood stem cells for unrelated hematopoietic stem cell transplantation for hematologic malignancies: on behalf of the Donor/Source Working Group of the Japanese Society for Transplantation and Cellular Therapy

Background aimsThis study aimed to comprehensively assess the impact of stem cell selection between bone marrow (BM) and peripheral blood (PB) in unrelated hematopoietic stem cell transplantation (HSCT) for hematological malignancies. Our objective was to identify specific factors associated with better transplant outcomes. MethodsA retrospective analysis was conducted using data from the Japanese HSCT registry. Inclusion criteria were patients aged 0–70 years who underwent their first unrelated HSCT with BM or PB, with an 8/8 or 7/8 allele HLA match for hematological malignancies between 2010 and 2020. ResultsAmong 10 295 patients, no significant difference was observed in overall survival, relapse, graft-versus-host disease (GVHD)-free, relapse-free survival (GRFS) or non-relapse mortality between the groups. Patients who received PB showed no clear difference in acute GVHD but had a greater rate of chronic GVHD, resulting in poor chronic GVHD-free, relapse-free survival (CRFS). Subgroup analyses highlighted the importance of patient-specific factors in source selection. Patients with non-Hodgkin lymphoma and a greater hematopoietic cell transplantation-comorbidity index showed better CRFS and GRFS when BM was the preferred source. Similar trends were observed among patients with standard-risk disease for CRFS. However, no such trends were evident among patients aged 0–24 years, indicating that both sources are viable choices for young patients. ConclusionsThis real-world retrospective analysis showed similar basic outcomes for BM and PB in an unrelated setting. The results support that BM may still be preferred over PB, especially when the long-term quality of life is a major concern. A consideration of individual factors can further optimize transplant success. Further research is warranted to explore the long-term implications of stem cell source selection.

Cellular Barcoding of JAK2-V617F Hematopoietic Stem Cells Reveals No Substantial Preferences in the Contribution of Individual Stem Cell Clones to Erythroid Versus Megakaryocytic Lineages

JAK2-V617F is the most frequent driver gene mutation in patients with myeloproliferative neoplasms (MPN) but the phenotypic manifestation is heterogeneous with some patients presenting as polycythemia vera (PV), whereas others with essential thrombocythemia (ET) or myelofibrosis (MF). JAK2-V617F is acquired in a hematopoietic stem cell (HSC) and leads to clonal expansion of HSCs and progenitor cells that gain dominance over unmutated hematopoiesis. We tested the hypothesis that the preferential expansion of megakaryopoiesis in ET versus erythropoiesis in PV could be due to the acquisition of JAK2-V617F in different subsets of HSCs with inherent bias towards megakaryopoiesis or erythropoiesis. In addition, we addressed the question of whether sensitivity or resistance to interferon-α (IFNα), currently the only treatment that can induce a deep molecular remission in some MPN patients, could be also due to heterogeneity in HSC subpopulations. We used a mouse model of JAK2-V617F driven MPN in combination with molecular barcoding that allowed us to monitor expansion and lineage contribution of individual HSC subclones. First, we genetically barcoded HSCs from our tamoxifen-inducible SclCre;JAK2-V617F ( VF) mice with a lentiviral vector, and transplanted 2'000 HSCs into lethally irradiated recipient mice. After 12 weeks we sequenced the barcodes in bone marrow (BM) progenitors by next generation sequencing (NGS). We observed a strong selection of 1-2 HSCs clones per recipient mouse, which alone contributed to &amp;gt; 80% of all the myeloid cells. Since cytokine storm after irradiation and aplasia during the reconstitution with lentivirally transduced BM may favor such oligo-clonal dominance observed in this experiment, we characterized the clonal composition of JAK2-mutant HSCs in non-transplanted MPN mice. To this end, we crossed a CRISPR-based genetic barcoding mouse line ( CARLIN) (Bowling, S. et al, Cell 2020) with our VF mice to obtain VF;CARLIN mice. Cas9-mediated barcoding in VF;CARLIN or in CARLIN mice with wildtype Jak2(WT;CARLIN) was induced with doxycycline and immediately followed by the activation of the JAK2-V617F by tamoxifen (Figure 1A). After 12 weeks, when the mice developed full PV phenotype (hemoglobin 195 g/L; platelets 4.3x10 12 /L and neutrophils 10.5x10 9/L), we performed scRNAseq on lin- cKit+ BM cells using the 10X-platform to retrieve the cellular barcodes and transcriptomic profiles. MPN hematopoiesis in these non-transplanted VF;CARLIN mice was polyclonal (Figure 1B). A total of &amp;gt; 500 barcoded clones per mouse were detected and in both VF;CARLIN and WT;CARLIN mice, but ~5 individual barcodes accounted for 25% of the cells in all progenitor subsets analyzed (Figure 1B). Overall, clonal composition was similar between WT;CARLIN and VF;CARLIN mice, indicating that clonal selection was not more prominent in JAK2-mutant mice. Importantly, in the offspring of HSCs that accounted for 25% of the cells, we did not observe preferential presence of some barcodes in MkP versus EryP and other barcodes in EryP versus MkP, arguing against the model predicting that erythrocytosis and thrombocytosis originated in different subsets of biased HSCs. To investigate the question of whether sensitivity or resistance to IFNα could be also due to heterogeneity in HSC subpopulations, we transplanted BM cells from these barcoded VF;CARLIN mice together with an excess of WT competitor cells into irradiated WT recipient mice, and treated them with IFNα or vehicle for 16 weeks. IFNα normalized hemoglobin (122 g/L) compared to vehicle (177 g/L) and reduced VF;CARLIN chimerism in granulocytes from 85% to 44%. BM cells were harvested and the clonal composition is currently being analyzed by NGS in order to identify IFNα resistant and sensitive subclones. Our barcoding analyses of individual HSCs showed that, during recovery after BM transplantations, a few VF clones dominated, whereas induction of VF expression in situ without transplantation resulted in a polyclonal disease that showed a similar number of HSCs actively contributing to hematopoiesis as in a control experiment with WT mice. The lack of substantial preference in the clonal contribution of individual HSCs to erythropoiesis versus megakaryopoiesis in VF mice suggests that PV and ET are not caused by acquiring JAK2-V617F in HSCs with a pre-existing lineage bias.

Arid1b Loss Impairs Hematopoietic Stem Cell Function in Normal Hematopoiesis

Switch Sugar Non- Fermenting (SWI/SNF) nucleosome remodeling complexes are multi subunits complexes that utilize the energy from ATP to remodel chromatin, affecting cellular processes on chromatin including that of gene transcription. SWI/SNF complexes play important roles in normal development and differentiation as well as in oncogenesis. ARID1 proteins, ARID1A and ARID1B, are unique and mutually exclusive subunits of the canonical SWI/SNF complex BRG-1/BRM Associated Factor (BAF) complex. Arid1a is the most frequently mutated SWI/SNF subunit in cancers and plays a vital role in hematopoiesis. Arid1b is also frequently mutated in cancers, and its loss promotes Acute Myeloid Leukemia leukemogenesis. However, its role in normal hematopoiesis is not known. In this study we used hematopoietic specific Cre strains, VavCre and Mx1Cre, to delete Arid1b in a mouse genetic model to study its role in normal hematopoiesis. Analysis of steady state hematopoiesis in the VavCre model showed no significant differences in complete blood count (CBC) between Arid1b fl/flVavCre+ and VavCre+ mice. Further, bone marrow (BM) cellularity was not significantly different between control and experimental mice. Flow cytometry analysis of the percentage of mature myeloid, B cell, or T cell populations in the peripheral blood (PB), BM, or spleen (SPL) did not show significant differences between Arid1b fl/flVavCre+ and VavCre+ control mice. Further, we did not observe any significant differences in percentages of hematopoietic stem and progenitor cells (HSPCs), including HSCs, common myeloid progenitors (CMPs), granulocyte macrophage progenitors (GMPs), megakaryocyte/erythrocyte progenitors (MEPs), and common lymphoid progenitors (CLPs) in the BM of Arid1b fl/flVavCre+ mice versus VavCre+ controls. Analysis of Mx1Cre model showed similar results. Taken together, these results suggest that Arid1b is dispensable for steady state hematopoiesis. To assess the effect of loss Arid1b in a regenerative setting, we conducted non-competitive and competitive transplantation experiments in VavCre and Mx1Cre models. We transplanted BM cells from Arid1b fl/flCre+ or Cre+ mice (CD45.2) into lethally irradiated recipient CD45.1 mice with (CD45.1 at 1:1 ratio) or without competitor cells for competitive or non-competitive transplantations respectively. In Mx1Cre model, deletion of Arid1b was induced 4 weeks after transplantation. We monitored donor chimerism in PB over time as well as in the BM and SPL at end point. In both VavCre and Mx1Cre non-competitive transplants, we did not observe significant differences in CBC or donor chimerism in mature cells between Arid1b fl/flCre+ and control Cre+ mice in the PB over time. Further, no significant differences were observed between knockout and control mice in donor chimerism in mature cell populations in the BM or SPL, nor HSPC populations in the BM at end point (24-28 weeks). Deletion of Arid1b in the BM was confirmed. These results indicate that Arid1b loss does not affect regenerative potential of HSPCs in a non-competitive setting. In our competitive transplants, we observed significant decreases in PB donor chimerism for both myeloid and B cell populations over time in Arid1b fl/flVavCre+ compared to VavCre+ controls. Further, we observed significant decreases in donor chimerism of mature myeloid cells and B cells in the BM and SPL, as well as HSCs, CMPs, GMPs, and MEPs in the BM of Arid1b fl/flVavCre+ mice compared to VavCre+ controls at endpoint. However, there seemed to be incomplete penetrance as the observed phenotype varied in cohorts of different donor mice and was not significant in all cohorts. A similar phenotype was observed in the Mx1Cre model with the exception of a trend, albeit insignificant, towards a decrease in donor derived mature B cell populations in Arid1b fl/flMx1Cre+ compared to Mx1Cre+ mice in PB, SPL, and BM and no significant difference in HSCs in the BM at end point (20 weeks). Deletion of Arid1b in the BM was confirmed in both models. Overall, our results suggest that Arid1b deficient HSCs can regenerate the hematopoietic system but have defects in competitive transplantations with incomplete penetrance. Further work is needed to address the observed incomplete penetrance phenotype in competitive transplantations, and to understand the mechanism behind the requirement of Arid1b in normal hematopoiesis.