Breaking barriers: supporting hematopoietic stem cell transplant program through collaborative radiation therapy service from a physically distant center.

BACKGROUND: Total body irradiation (TBI) for hematopoietic stem cell transplant (HSCT) has certain distinct advantages, such as uniform dose distribution and lack of drug resistance, but it is not widely available in resource-constrained settings. To overcome the limitations of in-house radiotherapy services in hematology centers, we evaluated the feasibility of conducting HSCT programs in coordination with two physically distant centers using a reduced-intensity TBI protocol. METHODS: Thirty-two patients with a median age of 20.5 years were included in the study. Fifteen patients were diagnosed with aplastic anemia, 10 patients with acute myeloid leukemia (AML), 3 patients with acute lymphocytic leukemia (ALL), and 4 patients with other hematological conditions. Conditioning regimens used were fludarabine plus cyclophosphamide in 29 cases, fludarabine-cytarabine ATG in 2 cases, and busulfan plus fludarabine in 1 case. The TBI dose was 3 Gy in 28 cases and 2 Gy in 4 cases. Patients were followed monthly after TBI, and the major toxicities were recorded. RESULTS: The median follow-up was 22 months. The most common acute complication was acute graft-versus-host disease (GVHD), which occurred in 15.6% of patients. The major late complications were chronic GVHD (9.3%), Cytomegalovirus (CMV) infection (34.3%), and CMV-induced secondary graft failure (6.2%). Seventy-five percent of patients were alive, 21.9% were dead, and 1 patient was lost to follow-up. CONCLUSIONS: HSCT based on TBI is feasible even if the center lacks a radiotherapy facility by coordinating with a remote radiotherapy facility. without compromising the patient's outcome.

[Efficacy of allogeneic hematopoietic stem cell transplantation based on a total body irradiation conditioning treatment regimen for adult acute lymphocytic leukemia].

Objective: To analyze the efficacy of allo-HSCT with total body irradiation (TBI) and chemotherapy alone in the treatment of adult ALL and to explore the factors affecting prognosis. Methods: The clinical data of 95 adult patients with ALL who underwent allo-HSCT from January 2015 to August 2022 were included. According to the conditioning regimen, the patients were divided into two groups: the TBI plus cyclophosphamide (TBI/Cy) group (n=53) and the busulfan plus cyclophosphamide (Bu/Cy) group (n=42). Hematopoietic reconstitution after transplantation, GVHD, transplantation-related complications, relapse rate (RR), non-relapse mortality (NRM), OS, and LFS were compared, and the factors related to prognosis were analyzed. Results: The median time of neutrophil engraftment was 14 (10-25) days in the TBI/Cy group and 14 (10-24) days in the Bu/Cy group (P=0.106). The median time of megakaryocyte engraftment was 17 (10-42) days in the TBI/Cy group and 19 (11-42) days in the Bu/Cy group (P=0.488). The incidence of grade Ⅱ-Ⅳ acute GVHD (aGVHD) in the TBI/Cy and Bu/Cy groups was 41.5% and 35.7%, respectively (P=0.565). The incidence of grade Ⅲ-Ⅳ aGVHD in these two groups was 24.5% and 4.8%, respectively (P=0.009). The incidence of severe chronic GVHD in the two groups was 16.7% and 13.5%, respectively (P=0.689). The incidence of cytomegalovirus infection, Epstein-Barr virus infection, severe infection, and hemorrhagic cystitis in the two groups was 41.5% and 35.7% (P=0.565), 34.0% and 35.7% (P=0.859), 43.4% and 33.3% (P=0.318), and 20.8% and 50.0% (P=0.003), respectively. The median follow-up time was 37.1 months and 53.3 months in the TBI/Cy and Bu/Cy groups, respectively. The 2-year cumulative RR was 17.0% in the TBI/Cy group and 42.9% in the Bu/Cy group (P=0.017). The 2-year cumulative NRM was 24.5% and 7.1%, respectively (P=0.120). The 2-year LFS was 58.5% and 50.0%, respectively (P=0.466). The 2-year OS rate was 69.8% and 64.3%, respectively (P=0.697). In the multivariate analysis, the conditioning regimen containing TBI was a protective factor for relapse after transplantation (HR=0.304, 95% CI 0.135-0.688, P=0.004), whereas the effect on NRM was not significant (HR=1.393, 95% CI 0.355-5.462, P=0.634). Infection was an independent risk factor for OS after allo-HSCT in adult patients with ALL. Conclusion: allo-HSCT based on TBI conditioning regimen had lower relapse rate and lower incidence of hemorrhagic cystitis for adult ALL, compared with chemotherapy regimen. While the incidence o grade Ⅲ/Ⅳ aGVHD was hgher in TBI conditioning regimen than that in chemotherapy regimen.

Dynamic equilibrium of skeletal muscle macrophage ontogeny in the diaphragm during homeostasis, injury, and recovery.

The diaphragm is a unique skeletal muscle due to its continuous activation pattern during the act of breathing. The ontogeny of macrophages, pivotal cells for skeletal muscle maintenance and regeneration, is primarily based on two distinct origins: postnatal bone marrow-derived monocytes and prenatal embryonic progenitors. Here we employed chimeric mice to study the dynamics of these two macrophage populations under different conditions. Traditional chimeric mice generated through whole body irradiation showed virtually complete elimination of the original tissue-resident macrophage pool. We then developed a novel method which employs lead shielding to protect the diaphragm tissue niche from irradiation. This allowed us to determine that up to almost half of tissue-resident macrophages in the diaphragm can be maintained independently from bone marrow-derived monocytes under steady-state conditions. These findings were confirmed by long-term (5 months) parabiosis experiments. Acute diaphragm injury shifted the macrophage balance toward an overwhelming predominance of bone marrow (monocyte)-derived macrophages. However, there was a remarkable reversion to the pre-injury ontological landscape after diaphragm muscle recovery. This diaphragm shielding method permits analysis of the dynamics of macrophage origin and corresponding function under different physiological and pathological conditions. It may be especially useful for studying diseases which are characterized by acute or chronic injury of the diaphragm and accompanying inflammation.

Thrombopoietin mimetic stimulates bone marrow vascular and stromal niches to mitigate acute radiation syndrome.

BACKGROUND: Acute radiation syndrome (ARS) manifests after exposure to high doses of radiation in the instances of radiologic accidents or incidents. Facilitating regeneration of the bone marrow (BM), namely the hematopoietic stem and progenitor cells (HSPCs), is key in mitigating ARS and multi-organ failure. JNJ-26366821, a PEGylated thrombopoietin mimetic (TPOm) peptide, has been shown as an effective medical countermeasure (MCM) to treat hematopoietic-ARS (H-ARS) in mice. However, the activity of TPOm on regulating BM vascular and stromal niches to support HSPC regeneration has yet to be elucidated. METHODS: C57BL/6J mice (9-14 weeks old) received sublethal or lethal total body irradiation (TBI), a model for H-ARS, by 137Cs or X-rays. At 24 h post-irradiation, mice were subcutaneously injected with a single dose of TPOm (0.3 mg/kg or 1.0 mg/kg) or PBS (vehicle). At homeostasis and on days 4, 7, 10, 14, 18, and 21 post-TBI with and without TPOm treatment, BM was harvested for histology, BM flow cytometry of HSPCs, endothelial (EC) and mesenchymal stromal cells (MSC), and whole-mount confocal microscopy. For survival, irradiated mice were monitored and weighed for 30 days. Lastly, BM triple negative cells (TNC; CD45-, TER-119-, CD31-) were sorted for single-cell RNA-sequencing to examine transcriptomics after TBI with or without TPOm treatment. RESULTS: At homeostasis, TPOm expanded the number of circulating platelets and HSPCs, ECs, and MSCs in the BM. Following sublethal TBI, TPOm improved BM architecture and promoted recovery of HSPCs, ECs, and MSCs. Furthermore, TPOm elevated VEGF-C levels in normal and irradiated mice. Following lethal irradiation, mice improved body weight recovery and 30-day survival when treated with TPOm after 137Cs and X-ray exposure. Additionally, TPOm reduced vascular dilation and permeability. Finally, single-cell RNA-seq analysis indicated that TPOm increased the expression of collagens in MSCs to enhance their interaction with other progenitors in BM and upregulated the regeneration pathway in MSCs. CONCLUSIONS: TPOm interacts with BM vascular and stromal niches to locally support hematopoietic reconstitution and systemically improve survival in mice after TBI. Therefore, this work warrants the development of TPOm as a potent radiation MCM for the treatment of ARS.

Ferroptosis, Inflammation, and Microbiome Alterations in the Intestine in the Göttingen Minipig Model of Hematopoietic-Acute Radiation Syndrome.

Hematopoietic acute radiation syndrome (H-ARS) involves injury to multiple organ systems following total body irradiation (TBI). Our laboratory demonstrated that captopril, an angiotensin-converting enzyme inhibitor, mitigates H-ARS in Göttingen minipigs, with improved survival and hematopoietic recovery, as well as the suppression of acute inflammation. However, the effects of captopril on the gastrointestinal (GI) system after TBI are not well known. We used a Göttingen minipig H-ARS model to investigate captopril's effects on the GI following TBI (60Co 1.79 or 1.80 Gy, 0.42-0.48 Gy/min), with endpoints at 6 or 35 days. The vehicle or captopril (0.96 mg/kg) was administered orally twice daily for 12 days, starting 4 h post-irradiation. Ilea were harvested for histological, protein, and RNA analyses. TBI increased congestion and mucosa erosion and hemorrhage, which were modulated by captopril. GPX-4 and SLC7A11 were downregulated post-irradiation, consistent with ferroptosis at 6 and 35 days post-irradiation in all groups. Interestingly, p21/waf1 increased at 6 days in vehicle-treated but not captopril-treated animals. An RT-qPCR analysis showed that radiation increased the gene expression of inflammatory cytokines IL1B, TNFA, CCL2, IL18, and CXCL8, and the inflammasome component NLRP3. Captopril suppressed radiation-induced IL1B and TNFA. Rectal microbiome analysis showed that 1 day of captopril treatment with radiation decreased overall diversity, with increased Proteobacteria phyla and Escherichia genera. By 6 days, captopril increased the relative abundance of Enterococcus, previously associated with improved H-ARS survival in mice. Our data suggest that captopril mitigates senescence, some inflammation, and microbiome alterations, but not ferroptosis markers in the intestine following TBI.

A novel AP/PA total body irradiation technique using abutting IMRT fields at extended SSD.

PURPOSE: To develop a Total Body Irradiation (TBI) technique using IMRT at extended SSD that can be performed in any size Linac room. METHODS: Patients studied were placed on a platform close to the floor, directly under the gantry with cranial-caudal axis parallel to the gantry rotation plane and at SSD ∼200 cm. Two abutting fields with the same external isocenter at gantry angles of ±21˚, collimator angle of 90˚, and field size of 25 × 40 cm2 are employed for both supine and prone positions. An iterative optimization algorithm was developed to generate a uniform dose at the patient mid-plane with adequate shielding to critical organs such as lungs and kidneys. The technique was validated in both phantom and patient CT images for treatment planning, and dose measurement and QA were performed in phantom. RESULTS: A uniform dose distribution in the mid-plane within ±5% of the prescription dose was reached after a few iterations. This was confirmed with ion-chamber measurements in phantom. The mean dose to lungs and kidneys can be adjusted according to clinical requirements and can be as low as ∼25% of the prescription dose. For a typical prescription dose of 200 cGy/fraction, the total MU was ∼2400/1200 for the superior/inferior field. The overall treatment time for both supine/prone positions was ∼54 min to meet the maximum absorbed dose rate criteria of 15 cGy/min. IMRT QA with portal dosimetry shows excellent agreement. CONCLUSIONS: We have developed a promising TBI technique using abutting IMRT fields at extended SSD. The patient is in a comfortable recumbent position with good reproducibility and less motion during treatment. An additional benefit of this technique is that full 3D dose distribution is available from the TPS with a DVH summary for organs of interest. The technique allows precise sparing of lungs and kidneys and can be executed in any linac room.

The paradigm of total body irradiation in acute lymphoblastic leukaemia: Therapeutic effectiveness versus the challenges of toxicity.

INTRODUCTION: Total body irradiation (TBI) is part of the myeloablative conditioning for hematopoietic stem cell transplantation (HSCT) in malignant hematologic disorders. This therapy has recently shown improved survival in acute lymphoblastic leukemia (ALL) compared to chemotherapy-based regimens. However, side effects are a significant limitation, especially in the pediatric population. PATIENTS AND METHODS: We retrospectively analyzed the survival of patients with ALL who underwent an HSCT at a tertiary hospital between 1996 and 2009 (N = 69 HSCT in 57 patients). We differentiated a cohort that received TBI (N = 44) from another that did not (N = 25). Subsequently, we interviewed the survivors from the TBI group with a minimum of 10 years of follow-up (N = 18), asking about the presence of side effects. RESULTS: The overall survival (OS) at 2 and 5 years was 79.1% and 65.2% respectively for the TBI group and 66.2% and 55.8% for the non-TBI group, although this difference was not significant (P=.31). The event-free survival (EFS) at 2 and 5 years was 77.3% and 63.6% respectively for the TBI group and 56% and 32% for the non-TBI group (P=.02). The probability of relapse (PR) at 2 years for those who received TBI was 10% compared to 28.6% for those who did not receive TBI (P=.005). Survivors who received TBI developed secondary neoplasms (39%), dyslipidemia (67%), cognitive impairments affecting memory (44%), recurrent respiratory infections (39%), thyroid abnormalities (45%), premature ovarian failure (89%), cataracts (22%), and psychological problems (44%). However, the quality of life, as self-assessed by the patients, was considered good for 83% of the participants.. CONCLUSIONS: Patients who received TBI had significantly higher EFS and lower PR. However, adverse effects are frequent and significant, although they do not subjectively affect quality of life.

Proteomic changes in the hippocampus of large mammals after total-body low dose radiation.

There is a growing interest in low dose radiation (LDR) to counteract neurodegeneration. However, LDR effects on normal brain have not been completely explored yet. Recent analyses showed that LDR exposure to normal brain tissue causes expression level changes of different proteins including neurodegeneration-associated proteins. We assessed the proteomic changes occurring in radiated vs. sham normal swine brains. Due to its involvement in various neurodegenerative processes, including those associated with cognitive changes after high dose radiation exposure, we focused on the hippocampus first. We observed significant proteomic changes in the hippocampus of radiated vs. sham swine after LDR (1.79Gy). Mass spectrometry results showed 190 up-regulated and 120 down-regulated proteins after LDR. Western blotting analyses confirmed increased levels of TPM1, TPM4, PCP4 and NPY (all proteins decreased in various neurodegenerative processes, with NPY and PCP4 known to be neuroprotective) in radiated vs. sham swine. These data support the use of LDR as a potential beneficial tool to interfere with neurodegenerative processes and perhaps other brain-related disorders, including behavioral disorders.

Alleviation of radiation combined skin injury in rat model by topical application of ascorbate formulation.

PURPOSE: This research endeavor was undertaken to elucidate the impact of an innovative ascorbate formulation on the regeneration process of full-thickness excision wounds in a rat model exposed to whole-body gamma irradiation, replicating conditions akin to combat or radiation emergency scenarios. MATERIALS AND METHODS: We established a comprehensive rat model by optimizing whole body γ-radiation doses (5-9 Gy) and full-thickness excision wound sizes (1-3 cm2) to mimic radiation combined injury (RCI). The developed RCI model was used to explore the healing potential of ascorbate formulation. The study includes various treatment groups (i.e., sham control, radiation alone, wound alone, radiation + wound, and radiation + wound + formulation). The ascorbate formulation was applied twice daily, with a 12-hour gap between each application, starting 1 hour after the initiation of the wound. The healing potential of the formulation in the RCI context was evaluated over 14 days through hematological, molecular, and histological parameters. RESULTS: The combination of a 5 Gy radiation dose and a 1 cm2 wound was identified as the optimal setting to develop the RCI model for subsequent studies. The formulation was used topically immediately following RCI, and then twice daily until complete healing. Treatment with the ascorbate formulation yielded noteworthy outcomes and led to a substantial reduction (p < .05) in the wound area, accelerated epithelialization periods, and an increased wound contraction rate. The formulation's localized healing response improved organ weights, normalized blood parameters, and enhanced hematopoietic and immune systems. A gene expression study revealed the treatment up-regulated TGF-ß and FGF, and down-regulated PDGF-α, TNF-α, IL-1ß, IL-6, MIP-1α, and MCP-1 (p < .05). Histopathological assessments supported the formulation's effectiveness in restoring cellular architecture and promoting tissue regeneration. CONCLUSION: Topical application of the ascorbate formulation in RCI resulted in a significant improvement in delayed wound healing, leading to accelerated wound closure by mitigating the expression of inflammatory responses.

Discovering the Radiation Biomarkers in the Plasma of Total-Body Irradiated Leukemia Patients.

The increased risk of acute large-scale radiological exposure for the world's population underlines the need for optimal radiation biomarkers. Ionizing radiation triggers a complex response by the genome, proteome, and metabolome, all of which have been reported as suitable indicators of radiation-induced damage in vivo. This study analyzed peripheral blood samples from total-body irradiation (TBI) leukemia patients through mass spectrometry (MS) to identify and quantify differentially regulated proteins in plasma before and after irradiation. In brief, samples were taken from 16 leukemic patients prior to and 24 h after TBI (2 × 2.0 Gy), processed with Tandem Mass Tag isobaric labelling kit (TMTpro-16-plex), and analyzed by MS. In parallel, label-free relative quantification was performed with a RP-nanoLC-ESI-MS/MS system in a Q-Exactive mass spectrometer. Protein identification was done in Proteome Discoverer v.2.2 platform (Thermo). Data is available via ProteomeXchange with identifier PXD043516. Using two different methods, we acquired two datasets of up-regulated (ratio ≥ 1.2) or down-regulated (ratio ≤ 0.83) plasmatic proteins 24 h after irradiation, identifying 356 and 346 proteins in the TMT-16plex and 285 and 308 label-free analyses, respectively (P ≤ 0.05). Combining the two datasets yielded 15 candidates with significant relation to gamma-radiation exposure. The majority of these proteins were associated with the inflammatory response and lipid metabolism. Subsequently, from these, five proteins showed the strongest potential as radiation biomarkers in humans (C-reactive protein, Alpha amylase 1A, Mannose-binding protein C, Phospholipid transfer protein, and Complement C5). These candidate biomarkers might have implications for practical biological dosimetry.

FSL-1: A Synthetic Peptide Increases Survival in a Murine Model of Hematopoietic Acute Radiation Syndrome.

In the current geopolitical climate there is an unmet need to identify and develop prophylactic radiation countermeasures, particularly to ensure the well-being of warfighters and first responders that may be required to perform on radiation-contaminated fields for operational or rescue missions. Currently, no countermeasures have been approved by the U.S. FDA for prophylactic administration. Here we report on the efficacious nature of FSL-1 (toll-like receptor 2/6 agonist) and the protection from acute radiation syndrome (ARS) in a murine total-body irradiation (TBI) model. A single dose of FSL-1 was administered subcutaneously in mice. The safety of the compound was assessed in non-irradiated animals, the efficacy of the compound was assessed in animals exposed to TBI in the AFRRI Co-60 facility, the dose of FSL-1 was optimized, and common hematological parameters [complete blood cell (CBC), cytokines, and bone marrow progenitor cells] were assessed. Animals were monitored up to 60 days after exposure and radiation-induced damage was evaluated. FSL-1 was shown to be non-toxic when administered to non-irradiated mice at doses up to 3 mg/kg. The window of efficacy was determined to be 24 h prior to 24 h after TBI. FSL-1 administration resulted in significantly increased survival when administered either 24 h prior to or 24 h after exposure to supralethal doses of TBI. The optimal dose of FSL-1 administration was determined to be 1.5 mg/kg when administered prior to irradiation. Finally, FSL-1 protected the hematopoietic system (recovery of CBC and bone marrow CFU). Taken together, the effects of increased survival and accelerated recovery of hematological parameters suggests that FSL-1 should be developed as a novel radiation countermeasure for soldiers and civilians, which can be used either before or after irradiation in the aftermath of a radiological or nuclear event.

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.

High variability in short and long-term recovery kinetic of blood cell count and blood chemistry in a partial body irradiation mouse model.

PURPOSE: In the aftermath of a nuclear disaster or accident, survivors will suffer from radiation-induced normal tissue damage. Recovery after radiation exposure is dictated by several factors, one of which is degree of shielding at time of exposure. This study aims to characterize the short and late term changes in kinetics and magnitude of pancytopenia and blood chemistry in a model of heterogeneous radiation exposure, or partial body irradiation (PBI), compared to whole body irradiation (WBI). MATERIALS AND METHODS: Male C57BL/6 mice, 8-10 weeks of age, were WBI at 6 different doses (6, 6.1. 6.15, 6.2, 6.5, and 7.5 Gy) to establish the LD50. To determine the effect of shielding on blood cell counts and chemistry, animals were either WBI at 6 Gy (LD2230) or 6 Gy PBI with one leg shielding (LD030). Complete blood counts and chemistry were measured at 1, 5-, 10-, 20-, 30- and 120-days post-irradiation. RESULTS AND CONCLUSIONS: Irradiated animals had significant depletion of white blood cells, red blood cells and platelets up to 10 days post-irradiation. Separation between PBI and WBI were observed at 10- and 20-days post-irradiation at which point PBI animals showed sign of recovery while overall cell count remains depleted in WBI animals up to 30 days post-irradiation. In addition, significant changes were found in parameters indicative of hematopoietic injury including hemoglobin count, hematocrit count and white blood cell population. Significant changes were observed in kidney function with changes to blood urea nitrogen and calcium concentration at 5-days post-irradiation. At 10-days post-irradiation. liver function changes differentiated WBI from PBI animals. Long-term, irradiated animal's chemistry values and many blood counts were not significantly different from Sham. In conclusion, partial shielding ensured complete survival and demonstrated a different recovery kinetics of blood and chemistry parameters after irradiation compared to survivors of whole body irradiation and no single hemopoietic parameter was able to consistently differentiate irradiated from Sham animals. This seems to indicate that there is no single robust hemopoietic parameter to differentiate those exposed from those who were not due to the inherent variability in individual responses. Furthermore, there were no significant long-term effects on these blood parameters between survivors of WBI and PBI except that shielding accelerated recovery.

Fluacrypyrim Protects Hematopoietic Stem and Progenitor Cells against Irradiation via Apoptosis Prevention.

Ionizing radiation (IR)-induced hematopoietic injury has become a global concern in the past decade. The underlying cause of this condition is a compromised hematopoietic reserve, and this kind of hematopoietic injury could result in infection or bleeding, in addition to lethal mishaps. Therefore, developing an effective treatment for this condition is imperative. Fluacrypyrim (FAPM) is a recognized effective inhibitor of STAT3, which exhibits anti-inflammation and anti-tumor effects in hematopoietic disorders. In this context, the present study aimed to determine whether FAPM could serve as a curative agent in hematopoietic-acute radiation syndrome (H-ARS) after total body irradiation (TBI). The results revealed that the peritoneally injection of FAPM could effectively promote mice survival after lethal dose irradiation. In addition, promising recovery of peripheral blood, bone marrow (BM) cell counts, hematopoietic stem cell (HSC) cellularity, BM colony-forming ability, and HSC reconstituting ability upon FAPM treatment after sublethal dose irradiation was noted. Furthermore, FAPM could reduce IR-induced apoptosis in hematopoietic stem and progenitor cells (HSPCs) both in vitro and in vivo. Specifically, FAPM could downregulate the expressions of p53-PUMA pathway target genes, such as Puma, Bax, and Noxa. These results suggested that FAPM played a protective role in IR-induced hematopoietic damage and that the possible underlying mechanism was the modulation of apoptotic activities in HSCs.

Empagliflozin attenuates radiation-induced hematopoietic damage via NOX-4/ROS/p38 pathway.

PURPOSE: Damage to the hematopoietic system and functional inhibition are severe consequences of radiation exposure. In this study, we have investigated the effect of empagliflozin on radiation-induced hematopoietic damage, with the aim of providing new preventive approach to such injuries. METHODS AND MATERIALS: Mice were given 4 Gy total body irradiation (TBI) 1 h after the oral administration of empagliflozin, followed by the continuous administration of the same dose of empagliflozin for 6d, and then sacrificed on the 10th day after irradiation. The reactive oxygen species (ROS) levels in hematopoietic cells and their regulatory mechanisms were also been investigated. Colony forming unit granulocyte macrophage assay and bone marrow transplantation assays were performed to detect the function of the bone marrow cells. KEY FINDINGS: Empagliflozin increased the cell viability, reduced ROS levels, and attenuated apoptosis in vitro after the bone marrow cells were exposed to 1 Gy radiation. Empagliflozin significantly attenuated ionizing radiation injuries to the hematopoietic system, increased the peripheral blood cell count, and enhanced the proportion and function of hematopoietic stem cells in mice exposed to 4 Gy TBI. These effects may be related to the NOX-4/ROS/p38 pathway-mediated suppression of MAPK in hematopoietic stem cells. Empagliflozin also influenced the expression of Nrf-2 and increased glutathione peroxidase activity, thereby promoting the clearance of reactive oxygen species. Furthermore, empagliflozin mitigated metabolic abnormalities by inhibiting the mammalian target of rapamycin. SIGNIFICANCE: Our study has demonstrated that empagliflozin can reduce radiation-induced injury in hematopoietic stem cells. This finding suggests that empagliflozin is a promising novel agent for preventing radiation-induced damage to the hematopoietic system.

Non-contact scintillator imaging dosimetry for total body irradiation in radiotherapy.

Objective.The goal of this work was to assess the potential use of non-contact scintillator imaging dosimetry for tracking delivery in total body irradiation (TBI).Approach. Studies were conducted to measure the time-gated light signals caused by radiation exposure to scintillators that were placed on tissue. The purpose was to assess efficacy in conditions common for TBI, such as the large source to surface distance (SSD) commonly used, the reduced dose rate, the inclusion of a plexiglass spoiler, angle of incidence and effects of peripheral patient support structures. Dose validation work was performed on phantoms that mimicked human tissue optical properties and body geometry. For this work, 1.5 cm diameter scintillating disks were developed and affixed to phantoms under various conditions. A time-gated camera synchronized to the linac pulses was used for imaging. Scintillation intensity was quantified in post processing and the values verified with simultaneous thermolumiescent dosimeter (TLD) measurements. Mean scintillation values in each region were compared to TLD measurements to produce dose response curves, and scatter effects from the spoiler and patient bed were quantified.Main results.The dose determined by scintillators placed in TBI conditions agreed with TLD dose determinations to within 2.7%, and did so repeatedly within 1.0% standard deviation variance. A linear fit between scintillator signal and TLD dose was achieved with anR2= 0.996 across several body sites. Scatter from the patient bed resulted in a maximum increase of 19% in dose.Significance.This work suggests that non-contact scintillator imaging dosimetry could be used to verify dose in real time to patients undergoing TBI at the prescribed long SSD and low dose rate. It also has shown that patient transport stretchers can significantly influence surface dose by increasing scatter.

Metabolomic Profiles in Tissues of Nonhuman Primates Exposed to Either Total- or Partial-Body Radiation.

A complex cascade of systemic and tissue-specific responses induced by exposure to ionizing radiation can lead to functional impairment over time in the surviving population. Current methods for management of survivors of unintentional radiation exposure episodes rely on monitoring individuals over time for the development of adverse clinical symptoms due to the lack of predictive biomarkers for tissue injury. In this study, we report on changes in metabolomic and lipidomic profiles in multiple tissues of nonhuman primates (NHPs) that received either 4.0 Gy or 5.8 Gy total-body irradiation (TBI) of 60Co gamma rays, and 4.0 or 5.8 Gy partial-body irradiation (PBI) from LINAC-derived photons and were treated with a promising radiation countermeasure, gamma-tocotrienol (GT3). These include small molecule alterations that correlate with radiation effects in the jejunum, lung, kidney, and spleen of animals that either survived or succumbed to radiation toxicities over a 30-day period. Radiation-induced metabolic changes in tissues were observed in animals exposed to both doses and types of radiation, but were partially alleviated in GT3-treated and irradiated animals, with lung and spleen being most responsive. The majority of the pathways protected by GT3 treatment in these tissues were related to glucose metabolism, inflammation, and aldarate metabolism, suggesting GT3 may exert radioprotective effects in part by sparing these pathways from radiation-induced dysregulation. Taken together, the results of our study demonstrate that the prophylactic administration of GT3 results in metabolic and lipidomic shifts that likely provide an overall advantage against radiation injury. This investigation is among the first to highlight the use of a molecular phenotyping approach in a highly translatable NHP model of partial- and total-body irradiation to determine the underlying physiological mechanisms involved in the radioprotective efficacy of GT3.

Validating Radiosensitivity with Pre-Exposure Differential Gene Expression in Peripheral Blood Predicting Survival and Non-Survival in a Second Irradiated Rhesus Macaque Cohort.

Radiosensitivity differs in humans and possibly in closely related nonhuman primates. The reasons for variation in radiosensitivity are not well known. In an earlier study, we examined gene expression (GE) pre-radiation in peripheral blood among male (n = 62) and female (n = 60) rhesus macaques (n = 122), which did or did not survive (up to 60 days) after whole-body exposure of 7.0 Gy (LD66/60). Eight genes (CHD5, CHI3L1, DYSF, EPX, IGF2BP1, LCN2, MBOAT4, SLC22A4) revealed significant associations with survival. Access to a second rhesus macaque cohort (males = 40, females = 23, total n = 63) irradiated with 5.8-7.2 Gy (LD29-50/60) and some treated with gamma-tocotrienol (GT3, a radiation countermeasure) allowed us to validate these gene expression changes independently. Total RNA was isolated from whole blood samples and examined by quantitative RT-PCR on a 96-well format. cycle threshold (Ct)-values normalized to 18S rRNA were analyzed for their association with survival. Regardless of the species-specific TaqMan assay, similar results were obtained. Two genes (CHD5 and CHI3L1) out of eight revealed a significant association with survival in the second cohort, while only CHD5 (involved in DNA damage response and proliferation control) showed mean gene expression changes in the same direction for both cohorts. No expected association of CHD5 GE with dose, treatment, or sex could be established. Instead, we observed significant associations for those comparisons comprising pre-exposure samples with CHD5 Ct values ≤ 11 (total n = 17). CHD5 Ct values ≤ 11 in these comparisons were mainly associated with increased frequencies (61-100%) of non-survivors, a trend which depending on the sample numbers, reached significance (P = 0.03) in males and, accordingly, in females. This was also reflected by a logistic regression model including all available samples from both cohorts comprising CHD5 measurements (n = 104, odds ratio 1.38, 95% CI 1.07-1.79, P = 0.01). However, this association was driven by males (odds ratio 1.62, 95% CI 1.10-2.38, P = 0.01) and CHD5 Ct values ≤ 11 since removing low CHD5 Ct values from this model, converted to insignificance (P = 0.19). A second male subcohort comprising high CHD5 Ct values ≥ 14.4 in both cohorts (n = 5) appeared associated with survival. Removing these high CHD5 Ct values converted the model borderline significant (P = 0.051). Based on the probability function of the receiver operating characteristics (ROC) curves, 8 (12.3%) and 5 (7.7%) from 65 pre-exposure RNA measurements in males, death and survival could be predicted with a negative and positive predictive value ranging between 85-100%. An associated odds ratio reflected a 62% elevated risk for dying or surviving per unit change (Ct-value) in gene expression, considering the before-mentioned CHD5 thresholds in RNA copy numbers. In conclusion, we identified two subsets of male animals characterized by increased (Ct values ≤ 11) and decreased (Ct values ≥ 14.4) CHD5 GE copy numbers before radiation exposure, which independently of the cohort, radiation exposure or treatment appeared to predict the death or survival in males.

Validation of genes for H-ARS severity prediction in leukemia patients - interspecies comparison, challenges, and promises.

PURPOSE: In a previous baboon-study, a total of 29 genes were identified for clinical outcome prediction of the hematologic, acute, radiation, syndrome (H-ARS) severity. Among them, four genes (FDXR, DDB2, POU2AF1, WNT3) appeared promising and were validated in five leukemia patients. Within this study, we sought further in-vivo validation in a larger number of whole-body irradiated patients. MATERIAL AND METHODS: Peripheral blood was drawn from 10 leukemia patients before and up to 3 days during a fractionated (2 Gy/day) total-body irradiation (TBI) with 2-12Gy. After RNA-isolation, gene expression (GE) was evaluated on 31 genes widely used in biodosimetry and H-ARS prediction employing qRT-PCR. A customized low-density-array (LDA) allowed simultanously analyzing all genes, the 96-well format further examined the four most promising genes. Fold-changes (FC) in GE relative to pre-irradiation were calculated. RESULTS: Five patients suffering from acute-lymphoblastic-leukemia (ALL) respectively non-Hodgkin-lymphoma (NHL) revealed sufficient RNA-amounts and corresponding lymphocyte and neutrophile counts for running qRT-PCR, while acute-myeloid-leukemia (AML) and one myelofibrosis patient could not supply enough RNA. Generally, 1-2µg total RNA was isolated, whereas up to 10-fold differences in RNA-quantities (associated suppressed GE-changes) were identified among pre-exposure and exposure samples. From 31 genes, 23 were expressed in at least one of the pre-exposure samples. Relative to pre-exposure, the number of expressed genes could halve at 48 and 72h after irradiation. Using the LDA, 13 genes were validated in human samples. The four most promising genes (vid. sup.) were either undetermined or too close to pre-exposure. However, they were measured using the more sensitive 96-well format, except WNT3, which wasn´t detectable. As in previous studies, an opposite regulation in GE for FDXR in leukemia patients (up-regulated) relative to baboons (down-regulated) was reconfirmed. Radiation-induced GE-changes of DDB2 (up-regulated) and POU2AF1 (down-regulated) behaved similarly in both species. Hence, 16 out of 23 genes of two species showed GE-changes in the same direction, and up-regulated FDXR as in human studies were revalidated. CONCLUSION: Identified genes for H-ARS severity prediction, previously detected in baboons, were validated in ALL but not in AML patients. Limitations related to leukemia type, associated reduced RNA amounts, suppressed GE changes, and methodological challenges must be considered as factors negatively affecting the total number of validated genes. Based on that, we propose additional controls including blood cell counts and preferably fluorescence-based RNA quantity measurements for selecting promising samples and using a more sensitive 96-well format for candidate genes with low baseline copy numbers.

Anti-radiation effect of MRN-100: a hydro-ferrate fluid, in vivo.

Ionizing radiation (IR) severely harms many organs, especially the hematopoietic tissue, mandating the development of protective nutraceuticals. MRN-100, a hydro-ferrate fluid, has been shown to protect γ-radiated fish against hematopoietic tissue damage and lethality. The current study aimed to examine MRN-100's protective effect against irradiated mice and explore the mechanisms underlying its effect. Mice received a single acute, sub-lethal, 5 Gy, whole body dose of X-ray IR. MRN-100 treatment was administered daily for 2-weeks pre-irradiation until 1-week post-irradiation. Spleen and blood were analysed for oxidative stress, hematological, histological and biochemical parameters. Radiation exposure markedly decreased complete blood count (CBC) parameters including hemoglobin, hematocrit, red blood cells, platelets, white blood cells and lymphocytes, and significantly increased neutrophils. In contrast, MRN-100 supplementation to irradiated mice ameliorated all CBC parameters and protected against DNA damage in both splenic cells and serum. It also had an antioxidant effect, increasing the levels of glutathione, superoxide dismutase, catalase and total antioxidant capacity, which were otherwise decreased by irradiation. MRN-100 intake reduced the oxidative stress biomarker levels of nitric oxide, protein carbonyl, malondialdehyde, reactive oxygen species and 8-hydroxydeoxyguanosine, a marker specific to DNA damage. Furthermore, MRN-100 enhanced serum iron and reversed the radiation-induced elevations of liver enzymes. Finally, MRN-100 protected splenic tissue from irradiation as observed by histology. We conclude that MRN-100 consumption may protect against oxidative stress generated by radiation exposure, suggesting that it may be employed as an adjuvant treatment to prevent radiation's severe damage to important organs.