To Total Body Irradiation Research Articles

Ionizing Radiation Dose Differentially Affects the Host-Microbe Relationship over Time.

Microorganisms that colonize in or on a host play significant roles in regulating the host's immunological fitness and bioenergy production, thus controlling the host's stress responses. Radiation elicits a pro-inflammatory and bioenergy-expensive state, which could influence the gut microbial compositions and, therefore, the host-microbe bidirectional relationship. To test this hypothesis, young adult mice were exposed to total body irradiation (TBI) at doses of 9.5 Gy and 11 Gy, respectively. The irradiated mice were euthanized on days 1, 3, and 9 post TBI, and their descending colon contents (DCCs) were collected. The 16S ribosomal RNAs from the DCCs were screened to find the differentially enriched bacterial taxa due to TBI. Subsequently, these data were analyzed to identify the metagenome-specific biofunctions. The bacterial community of the DCCs showed increased levels of diversity as time progressed following TBI. The abundance profile was the most divergent at day 9 post 11 Gy TBI. For instance, an anti-inflammatory and energy-harvesting bacterium, namely, Firmicutes, became highly abundant and co-expressed in the DCC with pro-inflammatory Deferribacteres at day 9 post 11 Gy TBI. A systems evaluation found a diverging trend in the regulation profiles of the functional networks that were linked to the bacteria and metabolites of the DCCs, respectively. Additionally, the network clusters associated with lipid metabolism and bioenergy synthesis were found to be activated in the DCC bacteria but inhibited in the metabolite space at day 9 post 11 Gy. Taking these results together, the present analysis indicated a disrupted mouse-bacteria symbiotic relationship as time progressed after lethal irradiation. This information can help develop precise interventions to ameliorate the symptoms triggered by TBI.

Biomarkers for Radiation Biodosimetry and Correlation with Hematopoietic Injury in a Humanized Mouse Model.

After a large-scale radiological or nuclear event, hundreds of thousands of people may be exposed to ionizing radiation and require subsequent medical management. Acute exposure to moderate doses (2-6 Gy) of radiation can lead to the hematopoietic acute radiation syndrome, in which the bone marrow (BM) is severely compromised, and severe hemorrhage and infection are common. Previously, we have developed a panel of intracellular protein markers (FDXR, ACTN1, DDB2, BAX, p53 and TSPYL2), designed to reconstruct absorbed radiation dose from human peripheral blood (PB) leukocyte samples in humanized mice up to 3 days after exposure. The objective of this work was to continue to use the humanized mouse model to evaluate biomarker dose-/time- kinetics in human PB leukocytes invivo, at an earlier (day 2) and later (day 7) time point, after exposure to total-body irradiation (TBI) doses of 0 to 2 Gy of X rays. In addition, to assess hematological sensitivity and radiation-induced injury, PB leukocyte cell counts, human BM hematopoietic stem cell (HSC) and progenitor cell [multipotent progenitor (MPP), common myeloid progenitor (CMP), granulocyte myeloid progenitor (GMP), megakaryocyte/erythrocyte progenitor (MEP) and multi-lymphoid progenitor (MLP)] levels were measured, and their correlation was also examined as the BM damages are difficult to assess by routine tests. Peripheral blood B-cells were significantly lower after TBI doses of 0.5 Gy on day 2 and 2 Gy on days 2 and 7; T-cells were significantly reduced only on day 2 after 2 Gy TBI. Bone marrow HSCs and MPP cells showed a dose-dependent depletion after irradiation with 0.5 Gy and 2 Gy on day 2, and after 1 Gy and 2 Gy on day 7. Circulating B cells correlated with HSCs, MPP and MLP cells on day 2, whereas T cells correlated with MPP, and myeloid cells correlated with MLP cells. On day 7, B cells correlated with MPP, CMP, GMP and MEP, while myeloid cells correlated with CMP, GMP and MEP. The intracellular leukocyte biomarkers were able to discriminate unirradiated and irradiated samples at different time points calculated by receiver operating characteristic (ROC) curve. Using machine learning algorithm methods, combining ACTN1, p53, TSPYL2 and PB-T cell and PB-B cell counts served as a strong predictor (area under the ROC >0.8) to distinguish unirradiated and irradiated samples independent of the days after TBI. The results further validated our biomarker-based triage assay and additionally evaluated the radiation sensitivity of the hematopoietic system after TBI exposures.

Time-restricted feeding prevents ionizing radiation-induced hematopoietic stem cell damage by inhibiting NOX-4/ROS/p38 MAPK pathway

Ionizing radiation (IR)-induced damage to the hematopoietic system is a prominent symptom following exposure to total body irradiation (TBI). The exploration of strategies aimed at to mitigating radiation-induced hematopoietic damage assumes paramount importance. Time-restricted feeding (TRF) has garnered attention for its beneficial effects in various diseases. In this study, we evaluated the preventive effects of TRF on TBI-induced hematopoietic damage. The results suggested that TRF significantly enhanced the proportion and function of hematopoietic stem cells in mice exposed to 4 Gy TBI. These effects might be attributed to the inhibition of the NOX-4/ROS/p38 MAPK pathway in hematopoietic stem cells. TRF also influenced the expression of nuclear factor erythroid2-related factor 2 and increased glutathione peroxidase activity, thereby promoting the clearance of reactive oxygen species. Furthermore, TRF alleviated aberrations in plasma metabolism by inhibiting the mammalian target of rapamycin. These findings suggest that TRF may represent a novel approach to preventing hematopoietic radiation damage.

Radioinduced Modulatory Response of Lipid Profiles of Wistar Rats Brain Monitored by DESI-MS Imaging

The ionizing radiation used in radiotherapy can break down deoxyribonucleic acid bonds, generate reactive oxygen and nitrogen species, alter intracellular proteins, and induce changes in the lipidome. This study delves into examining lipid classes and their spatial distribution in the brains of Wistar rats exposed to ionizing radiation. Healthy male isogenic Wistar rats (n = 15) were divided into a control group (CG, n = 3) and an irradiated group (IRG, n = 12). The individuals of the IRG group were subjected to total body irradiation (TBI) using a Co-60 source and a radiation dose of 5 Gy. The IRG group was subdivided into four subgroups corresponding to 168, 96, 48, and 24 h before sacrifice. The brains were sliced into the sagittal direction, and the slices were analyzed using the desorption electrospray ionization mass spectrometry imaging (DESI-MSI) approach. The mass spectrometry results and information from the Metabolite and Chemical Entity Database (METLINTM) and Lipid Metabolites and Pathways Strategy (LIPID MAPS) databases enabled the identification of lipids belonging to distinct classes. The DESI-MS images revealed distinctive spatial distributions of these lipids and the modulatory response induced by the radiation over time.

The Impact of CD45-Antibody-Drug Conjugate Conditioning on Clonal Dynamics and Immune Tolerance Post HSPC Transplantation in Rhesus Macaques

Compared to total body irradiation (TBI) or chemotherapy, antibody-drug conjugate (ADC) transplant conditioning may offer a more targeted and less toxic approach to clearing bone marrow niches to permit robust hematopoietic stem progenitor cell (HSPC) engraftment. Targeting the CD45 antigen, expressed on both HSPCs and immune cells, is of great interest for conditioning prior to both HSPC gene therapies and allo-transplantation, due to potential mitigation of immune rejection of neo or allo antigens. In this study, we investigated hematopoietic reconstitution and clonal dynamics following CD45-ADC (MGTA-45) conditioning and autologous transplantation of rhesus macaque HSPCs transduced with a barcoded lentiviral vector expressing the strong immunogen marker gene copGFP (GFP). Two macaques had mobilized HSPCs transduced with the barcoded GFP-expressing lentiviral vector, and then received a single dose of 0.2mg/kg CD45-ADC as sole pre-infusion conditioning. On day 10 following CD45-ADC, transduced autologous HSPCs were thawed and reinfused. Both animals developed expected cytopenias, requiring transient platelet and red blood cell transfusion support, but no other toxicities were observed. Engraftment defined as recovery of neutrophil counts to >500/µL and no further need for transfusions occurred promptly by day 13 post-transplantation for both animals. Both showed stable high level multilineage engraftment with GFP-expressing barcoded hematopoietic cells, at levels equivalent to or higher than previously achieved with busulfan chemotherapy (Fig. 1A). Myeloid cells showed higher initial marking levels in both animals than T lymphocytes, with a slow increase in marked T lymphocytes over time, likely due to the interval required for development of naïve T cells from HSPC in an adult thymus. Barcode clonal tracking in both animals revealed highly polyclonal and diverse HSPC engraftment, with lineage-restricted and transient clones contributing for the first 1-2 months (m), replaced by contributions from multilineage stable clones, similar to clonal dynamics we have reported following TBI or busulfan conditioning in the same macaque lentiviral barcoding model (Koelle et al, Blood, 2017; Abraham et al, MTCD, 2023). Clonal diversity was high by Shannon index (Fig. 1B), with very high cumulative clone numbers per animal of 28-34,000, comparable to prior animals conditioned with TBI or busulfan. Beginning at 8m post-transplantation in the first animal (H64K) and 4m in the second (HAWX), clonal diversity began to gradually drop (Fig. 1B), with loss of 63% in first monkey (11m compared to 4m) and 34% in second monkey (6m compared to 3.5m) of detected clones in granulocytes, coincident with a reduction in the fraction of GFP-expressing cells in all lineages and also in vector copy number as assessed by ddPCR, suggesting potential rejection of cells expressing xenogeneic copGFP. Anti-CopGFP antibodies were detected in the serum of both animals, arising coincident with decreases in circulating GFP-expressing cells. Flow cytometric phenotyping of serial samples demonstrated only partial depletion of blood and lymph node T cells at the 0.2mg/kg dose of CD45-ADC, despite potent marrow HSPC depletion, and may account for the immune rejection of cells expressing a foreign protein. However, the rejection of GFP-expressing cells was much slower and less complete than following busulfan conditioning, when autologous HSPCs labeled with the same vector were rejected completely by 4-6 weeks post transplantation, suggesting some degree of tolerance induced by CD45-ADC conditioning. Though long-term follow-up is needed to accurately compare ADC conditioning to TBI and busulfan, these findings underscore the potential of CD45-ADC conditioning as an efficacious, targeted and non-toxic approach to opening the bone marrow niche for genetically modified HSPCs. We are currently escalating the dose of CD45-ADC to achieve more profound T cell depletion at the time of transplantation, in hopes of producing lasting tolerance to vector-delivered transgenes or allogeneic HSPCs.

PEGylated thrombopoietin mimetic, JNJ‑26366821 a novel prophylactic radiation countermeasure for acute radiation injury

Thrombopoietin (TPO) is the primary regulator of platelet generation and a stimulator of multilineage hematopoietic recovery following exposure to total body irradiation (TBI). JNJ‑26366821, a novel PEGylated TPO mimetic peptide, stimulates platelet production without developing neutralizing antibodies or causing any adverse effects. Administration of a single dose of JNJ‑26366821 demonstrated its efficacy as a prophylactic countermeasure in various mouse strains (males CD2F1, C3H/HeN, and male and female C57BL/6J) exposed to Co-60 gamma TBI. A dose dependent survival efficacy of JNJ‑26366821 (− 24 h) was identified in male CD2F1 mice exposed to a supralethal dose of radiation. A single dose of JNJ‑26366821 administered 24, 12, or 2 h pre-radiation resulted in 100% survival from a lethal dose of TBI with a dose reduction factor of 1.36. There was significantly accelerated recovery from radiation-induced peripheral blood neutropenia and thrombocytopenia in animals pre-treated with JNJ‑26366821. The drug also increased bone marrow cellularity and megakaryocytes, accelerated multi-lineage hematopoietic recovery, and alleviated radiation-induced soluble markers of bone marrow aplasia and endothelial damage. These results indicate that JNJ‑26366821 is a promising prophylactic radiation countermeasure for hematopoietic acute radiation syndrome with a broad window for medical management in a radiological or nuclear event.

Survivors of Pediatric Hematopoietic Stem Cell Transplantation Exhibit Progressive Diastolic Dysfunction Over Years of Follow-Up

Patients who have undergone hematopoietic stem cell transplantation (HSCT) in childhood have a higher risk of diastolic heart failure (HF). The rate of progression of diastolic dysfunction in aging pediatric patients is unknown and is more difficult to assess in young patients secondary to changes in diastolic indices as they grow. HSCT recipients at our center were previously found to have decline in diastolic function indices at 1 year after HSCT. This study provides follow-up of this cohort, using age-normalized z-scores to assess whether the decline in diastolic function noted at 1-year post-HSCT persists, worsens, or improves over time. Patients age <21 years who underwent HSCT at Boston Children's Hospital/Dana-Farber Cancer Center between 2005 and 2008 with ≥3 surveillance echocardiograms, including 1 performed pre-HSCT, were included. Diastolic measures included mitral inflow (E/A ratio) and Doppler tissue imaging of left ventricular lateral wall (LV lateral e'), LV septal wall (septal e') and right ventricular free wall (RV e'). Systolic function was measured by LV ejection fraction (LVEF). Normalization by age was done using z-scores, and >±2 SD was defined as abnormal in linear modeling of diastolic dysfunction and systolic dysfunction over time. In a subset of patients with adequate post-HSCT images of the entire left atrium (LA), LA volume and LA strain analyses also were performed. The study cohort comprised 61 patients (41% female; median age at HSCT, 10.7 years; median follow-up, 7.4 years). Diastolic index z-scores declined by -.045/year for LV lateral e', -.06/year for LV septal e', and -.14/year for RV e' (P < .01). The E/A ratio z-score increased by .034/year (P=.028). Linear modeling demonstrated that LV lateral e' and LV septal e' would become abnormal at 25 and 20 years post-HSCT, respectively, whereas RV e' would become abnormal sooner, at 12.6 years. LVEF z-score declined by -.04/year (P < .01) and was estimated to become abnormal at 40 years post-HSCT. Exposure to total body irradiation (TBI) was associated with worsening diastolic indices, lower LVEF (P ≤ .002), and decreased LA reservoir strain (42.0% versus 45.0%; P=.016) and conduit strain (-31.5% versus -35.1%; P=.029), although there was significant overlap between TBI and anthracycline exposure. Treatment with anthracyclines even at low doses (median, 150 mg/m2) was associated with declining LVEF but not with changes in diastolic indices. Long-term survivors of childhood HSCT exhibit declines in both LV and RV diastolic function indices. These results inform the rate of progression of LV and RV diastolic dysfunction indices over time in long-term survivors of pediatric HSCT. A significant association was observed between TBI and diastolic dysfunction and a decline in LVEF. Treatment with anthracyclines even at low doses was associated with a mild decline in LVEF. Our results can inform a lifespan perspective on disease management in this population, encourage clinicians and patients to be vigilant in following guideline-directed surveillance echocardiography, and inform anticipatory responses by clinicians as patients transition from pediatric care to adult care.

Biomodified Extracellular Vesicles Remodel the Intestinal Microenvironment to Overcome Radiation Enteritis.

Ionizing radiation (IR) is associated with the occurrence of enteritis, and protecting the whole intestine from radiation-induced gut injury remains an unmet clinical need. Circulating extracellular vesicles (EVs) are proven to be vital factors in the establishment of tissue and cell microenvironments. In this study, we aimed to investigate a radioprotective strategy mediated by small EVs (exosomes) in the context of irradiation-induced intestinal injury. We found that exosomes derived from donor mice exposed to total body irradiation (TBI) could protect recipient mice against TBI-induced lethality and alleviate radiation-induced gastrointestinal (GI) tract toxicity. To enhance the protective effect of EVs, profilings of mouse and human exosomal microRNAs (miRNAs) were performed to identify the functional molecule in exosomes. We found that miRNA-142-5p was highly expressed in exosomes from both donor mice exposed to TBI and patients after radiotherapy (RT). Moreover, miR-142 protected intestinal epithelial cells from irradiation-induced apoptosis and death and mediated EV protection against radiation enteritis by ameliorating the intestinal microenvironment. Then, biomodification of EVs was accomplished via enhancing miR-142 expression and intestinal specificity of exosomes, and thus improved EV-mediated protection from radiation enteritis. Our findings provide an effective approach for protecting against GI syndrome in people exposed to irradiation.

1281: EXTRACELLULAR MITOCHONDRIA EXACERBATE GRAFT-VERSUS-HOST DISEASE MORTALITY IN A MOUSE MODEL

Introduction: Graft-versus-host disease (GVHD) is a major complication of hematopoietic stem cell transplantation (HSCT). Previous studies have shown an association between pre-transplant conditioning mediated tissue damage and incidence of GVHD, albeit the pathobiology is not clearly delineated. Recent studies have shown that tissue damage mediates release of extracellular mitochondria (exMito) and associated byproducts, which have immunomodulatory functions due to their archaebacterial origin. However, the role of exMito in the pathogenesis of GVHD has not been explored. Methods: Intestinal epithelial cells (Caco-2) were subjected to doses of irradiation used as pre-transplant conditioning. Subsequently, changes in intracellular and extracellular mitochondrial quantity and function were evaluated. BALB/c mice were subjected to total body irradiation (TBI) and plasma was analyzed to evaluate the quantity and function of circulating exMito. Finally, BALB/c mice undergoing a MHC-mismatched allogeneic HSCT were co-transplanted with exogenous isolated syngeneic mitochondria to study the impact of exMito on GVHD mortality. Results: Seahorse XF flux assays revealed that irradiation impaired intracellular mitochondrial function with a decrease in basal, ATP-linked and maximal respiratory capacity in Caco-2 cells. Analyses of the cell-culture supernatant showed elevated exMito and increased oxidative mitochondrial protein modification (carbonylation), following irradiation. BALB/c mice which received TBI demonstrated morphological signs of mitochondrial fission and damage in intestinal epithelium along with an increase in the amount of exMito in the plasma. In addition, exMito isolated from the plasma of irradiated animals had a markedly diminished response to pyruvate/malate-dependent-induction of mitochondrial membrane potential suggestive of damage. Finally, recipient mice co-transplanted with damaged cell-free mitochondria in a MHC-mismatched allogeneic HSCT demonstrated dose-dependent increased GVHD-induced mortality. Conclusions: The results indicate that conditioning regimen-mediated extracellular release of damaged mitochondria plays a role in GVHD mortality. Future studies targeting therapies to preserve mitochondrial function and decrease extracellular mitochondrial load for GVHD management is warranted.

Opaganib Protects against Radiation Toxicity: Implications for Homeland Security and Antitumor Radiotherapy.

Exposure to ionizing radiation (IR) is a lingering threat from accidental or terroristic nuclear events, but is also widely used in cancer therapy. In both cases, host inflammatory responses to IR damage normal tissue causing morbidity and possibly mortality to the victim/patient. Opaganib, a first-in-class inhibitor of sphingolipid metabolism, has broad anti-inflammatory and anticancer activity. Opaganib elevates ceramide and reduces sphingosine 1-phosphate (S1P) in cells, conditions that increase the antitumor efficacy of radiation while concomitantly suppressing inflammatory damage to normal tissue. Therefore, opaganib may suppress toxicity from unintended IR exposure and improve patient response to chemoradiation. To test these hypotheses, we first examined the effects of opaganib on the toxicity and antitumor activity of radiation in mice exposed to total body irradiation (TBI) or IR with partial bone marrow shielding. Oral treatment with opaganib 2 h before TBI shifted the LD75 from 9.5 Gy to 11.5 Gy, and provided substantial protection against gastrointestinal damage associated with suppression of radiation-induced elevations of S1P and TNFα in the small intestines. In the partially shielded model, opaganib provided dose-dependent survival advantages when administered 4 h before or 24 h after radiation exposure, and was particularly effective when given both prior to and following radiation. Relevant to cancer radiotherapy, opaganib decreased the sensitivity of IEC6 (non-transformed mouse intestinal epithelial) cells to radiation, while sensitizing PAN02 cells to in vitro radiation. Next, the in vivo effects of opaganib in combination with radiation were examined in a syngeneic tumor model consisting of C57BL/6 mice bearing xenografts of PAN02 pancreatic cancer cells and a cross-species xenograft model consisting of nude mice bearing xenografts of human FaDu cells. Mice were treated with opaganib and/or IR (plus cisplatin in the case of FaDu tumors). In both tumor models, the optimal suppression of tumor growth was attained by the combination of opaganib with IR (± cisplatin). Overall, opaganib substantially protects normal tissue from radiation damage that may occur through unintended exposure or cancer radiotherapy.

Endocrine complications after hematopoietic stem cell transplantation during childhood-Results from a close follow-up in a cohort of 152 patients.

Haematopoietic stem cell transplantation (HSCT) is a therapeutic option for numerous haematologic diseases and solid tumours. Increasing indications for HSCT and reduction in associated mortality have been raising the number of paediatric HSCT survivors and their long-term toxicities. To characterize the endocrine disorders developed after HSCT. Retrospective analysis of 152 patients submitted to HSCT in paediatric age with at least 24 months of follow-up at our endocrine late-effects clinics. Patients were followed up for 9.9 (interquartile range [IQR]: 12.2) years. The median age at HSCT was 7.5 (IQR: 9) years. At least one endocrine complication was observed in 65.1% of the patients. Primary hypogonadism was detected in 34.2%. Female gender (p < .001), HSCT > 10 years old (p = .01) and chemotherapy before HSCT (p < .001) were identified as risk factors for developing gonadal dysfunction. Growth hormone deficiency (GHD) occurred in 23.0% with a mean stature Z-score at diagnosis of -1.8 ± 1.4. GHD was associated with cranial (p < .001) and HSCT < 10 years old (p ≤ 0.001). Patients who were exposed to total body irradiation (TBI) were at higher risk for primary hypothyroidism (22.3%) (p = .01), thyroid nodules (17.1%) (p < .001), thyroid carcinoma (5.3%) (p < .001), dyslipidaemia (19.1%) (p < .001) and disturbance of carbohydrate metabolism (19.1%) (p < .001). At least one endocrine complication was diagnosed in 65.1% of patients, with gonadal dysfunction being the most prevalent. The conditioning regimen with TBI was a risk factor for the development of several endocrine disorders. This study is one of the largest series evaluating the endocrine disorders among survivors of paediatric HSCT and intends to reinforce the importance of routine follow-up of these patients.

Risk of Subsequent Malignant Neoplasms Following Hematopoietic Stem Cell Transplantation with Total Body Irradiation or Total Marrow Irradiation: Insights from Early Follow-Up

Total marrow irradiation (TMI) is an alternative to total body irradiation (TBI) as a component of the conditioning regimen for hematopoietic cell transplantation (HCT), offering the ability to deliver more targeted doses and facilitating organ-sparing. The organ-sparing effect of TMI is theorized to decrease the risk of complications associated with radiation, including subsequent malignant neoplasms (SMNs), while allowing for dosage escalation to improve oncologic outcomes. The purpose of this study was to compare SMNs rates among patients treated with TBI- or TMI-based conditioning regimens. We hypothesized that TMI would yield a rate of SMNs comparable to, if not lower than, TBI. A retrospective matched-pair analysis of patients who underwent allogeneic HCT and received either TBI- or TMI-based conditioning regimens to a total dose of 12 to 20 Gy was performed. A total of 171 patients received TMI-based conditioning and 171 received TBI-based conditioning, matched based on age, sex, diagnosis, and length of follow-up. SMNs were identified from an established long-term follow-up protocol, our institutional cancer registry, and the California Cancer Registry. There were no significant differences in patient and clinical characteristics between the TMI and TBI cohorts except for clinical response status at transplantation and radiation dose. As expected, patients in the TMI received higher radiation doses (median dose, 16.0 Gy for the TMI cohort versus 13.2 Gy for the TBI cohort; P < .001). The median follow-up for both cohorts was 2.0 years (range, .5 to 12.3 years). There was no significant difference in the risk of developing SMNs between the 2 cohorts (P=.81). A total of 9 patients (5.3%) conditioned with TBI and 10 patients (5.8%) conditioned with TMI developed SMNs, at a median of 3.3 years and 1.7 years following HCT, respectively. Excluding nonmelanoma skin cancers and noninvasive neoplasms, 2 patients in the TBI cohort developed SMNs (both melanomas), and 1 patient in the TMI cohort developed an SMN (colon cancer). No patients developed a subsequent hematologic malignancy. TMI-based conditioning is not associated with a significant difference in the risk of developing SMNs compared with TBI-based conditioning during early post-HCT follow-up. Future studies with longer follow-up may be needed to further characterize the risk of SMNs associated with TMI-based conditioning regimens compared with TBI-based regimens.

Haptoglobin is an early indicator of survival after radiation-induced severe injury and bone marrow transplantation in mice

BackgroundHematopoietic stem cell transplantation (HSCT) is the main treatment for acute radiation sickness, especially after fatal radiation. The determination of HSCT for radiation patients is mainly based on radiation dose, hemogram and bone marrow injury severity. This study aims to explore a better biomarker of acute radiation injury from the perspective of systemic immune response.MethodsC57BL/6J female mice were exposed to total body irradiation (TBI) and partial body irradiation (PBI). Changes in haptoglobin (Hp) level in plasma were shown at different doses and time points after the exposure and treatment with amifostine or bone marrow transplantation. Student’s t-test/two tailed test were used in two groups. To decide the Hp levels as a predictor of the radiation dose in TBI and PBI, multiple linear regression analysis were performed. The ability of biomarkers to identify two groups of different samples was determined by the receiver operating characteristic (ROC) curve. The results were expressed as mean ± standard deviation (SD). Significance was set at P value < 0.05, and P value < 0.01 was set as highly significant. Survival distribution was determined by log-rank test.ResultsIn this study, we found that Hp was elevated dose-dependently in plasma in the early post-irradiation period and decreased on the second day, which can be used as a molecular indicator for early dose assessment. Moreover, we detected the second increase of Hp on the 3rd and 5th days after the lethal irradiation at 10 Gy, which was eliminated by amifostine, a radiation protection drug, while protected mice from death. Most importantly, bone marrow transplantation (BMT) on the 3rd and 5th day after 10 Gy radiation improved the 30-days survival rate, and effectively accelerated the regression of secondary increased Hp level.ConclusionsOur study suggests that Hp can be used not only as an early molecule marker of radiation injury, but also as an important indicator of bone marrow transplantation therapy for radiation injury, bringing new scientific discoveries in the diagnosis and treatment of acute radiation injury from the perspective of systemic immunity.

Therapeutic Potential of Human Immature Dental Pulp Stem Cells Observed in Mouse Model for Acquired Aplastic Anemia.

Aplastic anemia (AA) is a rare and serious disorder of hematopoietic stem cells (HSCs) that results in the loss of blood cells due to the failure of the bone marrow (BM). Although BM transplantation is used to treat AA, its use is limited by donor availability. In this sense, mesenchymal stem cells (MSCs) can offer a novel therapeutic approach for AA. This is because the MSCs contribute to the hematopoietic niche organization through their repopulating. In our study, we used the human immature dental pulp stem cell (hIDPSC), an MSC-like cell, to explore an alternative therapeutic approach for AA. For this, isogenic C57BL/6 mice were exposed to total body irradiation (TBI) to induce the AA. After 48 h of TBI, the mice were intraperitoneally treated with hIDPSC. The immunohistochemistry analyses confirmed that the hIDPSCs migrated and grafted in the mouse bone marrow (BM) and spleen, providing rapid support to hematopoiesis recovery compared to the group exposed to radiation, but not to those treated with the cells as well as the hematological parameters. Six months after the last hIDPSC transplantation, the BM showed long-term stable hematopoiesis. Our data highlight the therapeutic plasticity and hematoprotective role of hIDPSC for AA and potentially for other hematopoietic failures.

Hematopoietic stem cell regeneration through paracrine regulation of the Wnt5a/Prox1 signaling axis.

The crosstalk between the BM microenvironment (niche) and hematopoietic stem cells (HSCs) is critical for HSC regeneration. Here, we show that in mice, deletion of the Fanconi anemia (FA) genes Fanca and Fancc dampened HSC regeneration through direct effects on HSCs and indirect effects on BM niche cells. FA HSCs showed persistent upregulation of the Wnt target Prox1 in response to total body irradiation (TBI). Accordingly, lineage-specific deletion of Prox1 improved long-term repopulation of the irradiated FA HSCs. Forced expression of Prox1 in WT HSCs mimicked the defective repopulation phenotype of FA HSCs. WT mice but not FA mice showed significant induction by TBI of BM stromal Wnt5a protein. Mechanistically, FA proteins regulated stromal Wnt5a expression, possibly through modulating the Wnt5a transcription activator Pax2. Wnt5a treatment of irradiated FA mice enhanced HSC regeneration. Conversely, Wnt5a neutralization inhibited HSC regeneration after TBI. Wnt5a secreted by LepR+CXCL12+ BM stromal cells inhibited β-catenin accumulation, thereby repressing Prox1 transcription in irradiated HSCs. The detrimental effect of deregulated Wnt5a/Prox1 signaling on HSC regeneration was also observed in patients with FA and aged mice. Irradiation induced upregulation of Prox1 in the HSCs of aged mice, and deletion of Prox1 in aged HSCs improved HSC regeneration. Treatment of aged mice with Wnt5a enhanced hematopoietic repopulation. Collectively, these findings identified the paracrine Wnt5a/Prox1 signaling axis as a regulator of HSC regeneration under conditions of injury and aging.

Peripheral blood parameter abnormalities precede therapy-related myeloid neoplasms after autologous transplantation for lymphoma.

Therapy-related myeloid neoplasms (t-MN) are a leading cause of nonrelapse mortality after autologous peripheral blood stem cell transplantation (aPBSCT) in patients with Hodgkin lymphoma (HL) and non-Hodgkin lymphomas (NHL). t-MN patients treated at an earlier stage of disease evolution have a better prognosis, and this presents a need to identify patients at risk for t-MN. Using a prospective longitudinal study design, this study evaluated peripheral blood parameters pre-aPBSCT and on day 100, at 6 months, 1 year, 2 years, and 3 years in 304 patients treated with aPBSCT. The relation between peripheral blood parameters and subsequent development of t-MN was examined, and nomograms were developed to identify patients at risk for t-MN. Twenty-one patients developed t-MN at a median of 1.95 years post-aPBSCT. Hemoglobin, hematocrit, white blood cell, and platelet counts were lower among patients who developed t-MN compared to those who did not; these differences appeared soon after aPBSCT, persisted, and preceded development of t-MN. Older age at aPBSCT (hazard ratio [HR]per_year_increase = 1.08, P = .007), exposure to total body irradiation (TBI) (HR = 2.90, P = .04), and low 100-day platelet count (HRincrease_per_unit_decline_in_PLT = 1.01, P = .002) predicted subsequent t-MN. These parameters and primary diagnosis allowed identification of patients at high risk of t-MN (eg, an HL patient undergoing aPBSCT at the age of 70 years with TBI and with a day 100 PLT between 100,000 and 150,000 would have a 62% probability of developing t-MN at 6 years post-aPBSCT). Abnormalities in peripheral blood parameters can identify patients at high risk for t-MN after aPBSCT for HL or NHL, allowing opportunities to personalize close surveillance and possible disease-modifying interventions.

Hematopoietic Stem Cell Regeneration through Paracrine Regulation of the Wnt5a-Prox1 Signaling Axis

The crosstalk between bone marrow (BM) microenvironment (niche) and hematopoietic stem cells (HSCs) is critical for HSC regeneration after injury. Here we show that deletion of the genes encoding the DNA repair-deficient syndrome Fanconi anemia (FA), Fanca and Fancc, in mice dampens HSC regeneration through both direct effects on HSCs and indirect effects on BM niche cells. Specifically, Fanca- or Fancc-deficiency compromises hematologic recovery and dampens HSC regeneration following irradiation. FA HSCs show persistent upregulation of the Wnt target Prox1, a homeobox transcription factor, in response to total body irradiation (TBI). Accordingly, lineage-specific deletion of Prox1 improves long-term repopulation of the irradiated FA HSCs. Forced expression of Prox1 in wild-type (WT) HSC mimics the defective repopulation phenotype of FA HSCs. By analyzing paracrine factors in Wnt signaling, we found that WT mice, but not FA mice, show significant induction by TBI of BM stromal Wnt5a protein, which is produced in LepR +CXCL12 + BM stromal cells. Wnt5a treatment of irradiated FA mice enhances hematopoietic recovery and HSC regeneration. Conversely, Wnt5a neutralization in co-cultured LepR + BM stromal cells inhibits HSC regeneration and hematopoietic recovery following TBI. Mechanistically, Wnt5a secreted by LepR +CXCL12 + BM stromal cells inhibits b-catenin accumulation, thereby repressing Prox1 transcription in irradiated HSPCs. The detrimental effect of deregulated Wnt5a-Prox1 signaling on HSC regeneration and hematopoietic recovery is also observed in aged mice. Irradiation induces upregulation of Prox1 in the HSCs of aged mice, and deletion of Prox1 in aged HSCs improves HSC regeneration and hematopoietic recovery after irradiation. Finally, treatment of aged mice with Wnt5a enhances hematopoietic repopulation. Collectively, these findings identify the novel paracrine Wnt5a-Prox1 signaling axis in regulating HSC regeneration under conditions of injury and aging. DisclosuresNo relevant conflicts of interest to declare.

Outcomes of pediatric patients with therapy-related myeloid neoplasms.

Long-term outcomes after allogeneic hematopoietic cell transplantation (HCT) for therapy-related myeloid neoplasms (tMNs) are dismal. There are few multicenter studies defining prognostic factors in pediatric patients with tMNs. We have accumulated the largest cohort of pediatric patients who have undergone HCT for a tMN to perform a multivariate analysis defining factors predictive of long-term survival. Sixty-eight percent of the 401 patients underwent HCT using a myeloablative conditioning (MAC) regimen, but there were no statistically significant differences in the overall survival (OS), event-free survival (EFS), or cumulative incidence of relapse and non-relapse mortality based on the conditioning intensity. Among the recipients of MAC regimens, 38.4% of deaths were from treatment-related causes, especially acute graft versus host disease (GVHD) and end-organ failure, as compared to only 20.9% of deaths in the reduced-intensity conditioning (RIC) cohort. Exposure to total body irradiation (TBI) during conditioning and experiencing grade III/IV acute GVHD was associated with worse OS. In addition, a diagnosis of therapy-related myelodysplastic syndrome and having a structurally complex karyotype at tMN diagnosis were associated with worse EFS. Reduced-toxicity (but not reduced-intensity) regimens might help to decrease relapse while limiting mortality associated with TBI-based HCT conditioning in pediatric patients with tMNs.

Caloric restriction alleviates radiation injuries in a sex-dependent fashion.

Safe and effective regimens are still needed given the risk of radiation toxicity from iatrogenic irradiation. The gut microbiota plays an important role in radiation damage. Diet has emerged as a key determinant of the intestinal microbiome signature and function. In this report, we investigated whether a 30% caloric restriction (CR) diet may ameliorate radiation enteritis and hematopoietic toxicity. Experimental mice were either fed ad libitum (AL) or subjected to CR preconditioning for 10days and then exposed to total body irradiation (TBI) or total abdominal irradiation (TAI). Gross examinations showed that short-term CR pretreatment restored hematogenic organs and improved the intestinal architecture in both male and female mice. Intriguingly, CR preconditioning mitigated radiation-induced systemic and enteric inflammation in female mice, while gut barrier function improved in irradiated males. 16S rRNA high-throughput sequencing showed that the frequency of pro-inflammatory microbes, including Helicobacter and Desulfovibrionaceae, was reduced in female mice after 10days of CR preconditioning, while an enrichment of short-chain fatty acid (SCFA)-producing bacteria, such as Faecalibaculum, Clostridiales, and Lactobacillus, was observed in males. Using fecal microbiota transplantation (FMT) or antibiotic administration to alter the gut microbiota counteracted the short-term CR-elicited radiation tolerance of both male and female mice, further indicating that the radioprotection of a 30% CR diet depends on altering the gut microbiota. Together, our findings provide new insights into CR in clinical applications and indicate that a short-term CR diet prior to radiation modulates sex-specific gut microbiota configurations, protecting male and female mice against the side effects caused by radiation challenge.

First Multimodal, Three-Dimensional, Image-Guided Total Marrow Irradiation Model for Preclinical Bone Marrow Transplantation Studies

Total marrow irradiation(TMI) has significantly advanced radiation conditioning for hematopoietic cell transplantation inhematologicmalignancies by reducing conditioning-induced toxicities and improving survival outcomes in relapsed/refractory patients. However, the relapse rate remains high, and the lack of a preclinical TMI model has hindered scientific advancements. To accelerate TMI translation to the clinic, we developed a TMI delivery system in preclinical models. A Precision X-RAD SmART irradiator was used for TMI model development. Images acquired with whole-body contrast-enhanced computed tomography (CT) were used to reconstruct and delineate targets and vital organs for each mouse. Multiple beam and CT-guided Monte Carlo-based plans were performed to optimize doses to the targets and to vary doses to the vital organs. Long-term engraftment and reconstitution potential were evaluated by a congenic bone marrow transplantation (BMT) model and serial secondary BMT, respectively. Donor cell engraftment was measured using noninvasive bioluminescence imaging and flow cytometry. Multimodal imaging enabled identification of targets (skeleton and spleen) and vital organs (eg, lungs, gut, liver). In contrast to total body irradiation (TBI), TMI treatment allowed variation of radiation dose exposure to organs relative to the target dose. Dose reduction mirrored that in clinical TMI studies. Similar to TBI, mice treated with different TMI regimens showed full long-term donor engraftment in primary BMT and second serial BMT. The TBI-treated mice showed acute gut damage, which was minimized in mice treated with TMI. A novel multimodal image guided preclinical TMI model is reported here. TMI conditioning maintained long-term engraftment with reconstitution potential and reduced organ damage. Therefore, this TMI model provides a unique opportunity to study the therapeutic benefit of reduced organ damage and BM dose escalation to optimize treatment regimens in BMT and hematologic malignancies.