Enzyme-linked ImmunoSpot (ELISpot) assay to quantify peptide-specific IFN-γ production by splenocytes in a mouse tumor model after radiation therapy.

To develop new effective therapeutic strategies for cancer patients, there is a need for extensive and precise insights into the mechanisms involved in the immune response to anti-cancer treatments. The enzyme-linked immunospot (ELISpot) assay is a rapid and reproducible technique that allows the detection of cytokine-producing antigen-specific T cells at the single cell level. This protocol describes an interferon gamma (IFN-γ) ELISpot method for measuring antigen-specific murine CD8+ T cells that produce IFN-γ, a marker of their activation and cytotoxicity. Splenocytes from tumor-bearing mice treated with radiation therapy were used as source of CD8+ T cells and were stimulated with a tumor-derived peptide. This method was facilitated by a ready-to-use assay kit and provides a tool to analyze the specificity, intensity, and kinetics of specific CD8+ T cells.

Treatments affecting splenic function as a risk factor for valvular heart disease in Childhood Cancer Survivors: A DCCSS-LATER study.

PURPOSE: Splenectomy might be a risk factor for valvular heart disease (VHD) in adult Hodgkin lymphoma survivors. As this risk is still unclear for childhood cancer survivors (CCS), the aim of this study is to evaluate the association between treatments affecting splenic function (splenectomy and radiotherapy involving the spleen) and VHD in CCS. METHODS: CCS were enrolled from the DCCSS-LATER cohort, consisting of 6,165 five-year CCS diagnosed between 1963 and 2002. Symptomatic VHD, defined as symptoms combined with a diagnostic test indicating VHD, was assessed from questionnaires and validated using medical records. Differences in the cumulative incidence of VHD between CCS who received treatments affecting splenic function and CCS who did not were assessed using the Gray test. Risk factors were analyzed in a multivariable Cox proportional hazards model. RESULTS: The study population consisted of 5,286 CCS, with a median follow-up of 22 years (5-50 years), of whom 59 (1.1%) had a splenectomy and 489 (9.2%) radiotherapy involving the spleen. VHD was present in 21 CCS (0.4%). The cumulative incidence of VHD at the age of 40 years was significantly higher in CCS who received treatments affecting splenic function (2.7%, 95% confidence interval (CI) 0.4%-4.9%) compared with CCS without (0.4%, 95% CI 0.1%-0.7%) (Gray's test, p = 0.003). Splenectomy was significantly associated with VHD in a multivariable analysis (hazard ratio 8.6, 95% CI 3.1-24.1). CONCLUSIONS AND IMPLICATIONS: Splenectomy was associated with VHD. Future research is needed to determine if CCS who had a splenectomy as part of cancer treatment might benefit from screening for VHD.

Spleen Volume Reduction Is a Reliable and Independent Biomarker for Long-Term Risk of Leukopenia Development in Peptide Receptor Radionuclide Therapy.

177Lu-DOTATATE therapy is an effective treatment for advanced neuroendocrine tumors, despite its dose-limiting hematotoxicity. Herein, the significance of off-target splenic irradiation is unknown. Our study aims to identify predictive markers of peptide receptor radionuclide therapy-induced leukopenia. Methods: We retrospectively analyzed blood counts and imaging data of 88 patients with histologically confirmed, unresectable metastatic neuroendocrine tumors who received 177Lu-DOTATATE treatment at our institution from February 2009 to July 2021. Inclusion criterium was a tumor uptake equivalent to or greater than that in the liver on baseline receptor imaging. We excluded patients with less than 24 mo of follow-up and those patients who received fewer than 4 treatment cycles, additional therapies, or blood transfusions during follow-up. Results: Our study revealed absolute and relative white blood cell counts and relative spleen volume reduction as independent predictors of radiation-induced leukopenia at 24 mo. However, a 30% decline in spleen volume 12 mo after treatment most accurately predicted patients proceeding to leukopenia at 24 mo (receiver operating characteristic area under the curve of 0.91, sensitivity of 0.93, and specificity of 0.90), outperforming all other parameters by far. Conclusion: Automated splenic volume assessments demonstrated superior predictive capabilities for the development of leukopenia in patients undergoing 177Lu-DOTATATE treatment compared with conventional laboratory parameters. The reduction in spleen size proves to be a valuable, routinely available, and quantitative imaging-based biomarker for predicting radiation-induced leukopenia. This suggests potential clinical applications for risk assessment and management.

Citicoline modulates inflammatory signaling pathways in the spleen of rats exposed to gamma-radiation.

BACKGROUND AND AIM: The spleen has an essential role in immune responses regulation and is considered the biggest peripheral immune organ. Citicoline is used for various brain disorders management. This study aimed to examine the using possibility of citicoline to treat γ-radiation-induced splenic inflammation in rats. MATERIALS AND METHODS: Eighteen male albino rats were classified into: Group 1 (control) animals were kept as control. Group 2 (γ-radiation) animals were total-body γ-irradiated with 6 Gy. Group 3 (γ-radiation + citicoline) rats were γ-irradiated with 6 Gy, then injected intraperitoneally with citicoline (300 mg/kg/d) 5 min after irradiation for one week. Levels of TNF-α, IL-1ß, iNOS, NF-κB, JAK2, and STAT3 were determined in spleen tissue, along with histopathological examination. RESULTS: Rats exposure to gamma-radiation led to elevation in splenic TNF-α, IL-1ß, NF-κB, iNOS, JAK2, and STAT3 levels significantly. Treatment with citicoline after gamma-radiation exposure improved this elevation, and modulated gamma-radiation-induced histopathological alterations. CONCLUSIONS: This data showed that citicoline inhibited γ-radiation-induced splenic inflammation via suppressing NF-κB and JAK2/STAT3 signaling pathways in spleen tissue.

Effect of Transplanted Bone Marrow on Spleen of Irradiated Pregnant Rats and Their Fetuses.

<b>Background and Objective:</b> Prenatal ionizing radiation exposure may hinder fetal and embryonic growth depending on the dose and gestational age. The current study's objective was to discover how bone marrow transplants affected the spleens of pregnant rats that had been subjected to γ (Gamma) radiation. <b>Materials and Methods:</b> Sixty rats that were pregnant were separated into five different groups, each with 6 females. The pregnant rats in the second Group were exposed to 2Gy of γ-rays. Group III; pregnant rats subjected to 2Gy of γ-rays, followed by an intraperitoneal injection of newly prepared bone marrow transplantation (BMT). The fifth Group were exposed to 2Gy γ-rays and received 1 dosage of BMT an hour later. Spleen samples from the pregnant rats as well as their fetuses were taken for histological and histochemical analyses. <b>Results:</b> Gamma rays damaged the splenic tissue of women and their fetuses on days 7 or 14 of pregnancy in a variety of histological and histochemical ways, although bone marrow transplantation significantly reduced the damage. Treated mothers with bone marrow post-radiation showed a noticeable recovery in spleen of their fetuses. Improved spleen architecture was accompanied by appearance of normal content of collagen, polysaccharides and total protein in the fetal spleen tissue especially on day 7 of gestation. <b>Conclusion:</b> Bone marrow transplantation can lessen the damage caused by gamma radiation.

The relationship between splenic dose and radiation-induced lymphopenia.

Lymphocytes, which are highly sensitive to radiation, play a crucial role in the body's defense against tumors. Radiation-induced lymphopenia has been associated with poorer outcomes in different cancer types. Despite being the largest secondary lymphoid organ, the spleen has not been officially designated as an organ at risk. This study hypothesizes a connection between spleen irradiation and lymphopenia and seeks to establish evidence-based dosage limits for the spleen. We retrospectively analyzed data from 96 patients with locally advanced gastric cancer who received postoperative chemoradiotherapy (CRT) between May 2010 and May 2017. Complete blood counts were collected before, during and after CRT. We established a model for predicting the minimum absolute lymphocyte count (Min ALC) and to investigate potential associations between spleen dosimetric variables and Min ALC. The median follow-up was 60 months. The 5-year overall survival (OS) and disease-free survival (DFS) were 65.2% and 56.8%, respectively. The median values of pre-treatment ALC, Min ALC and post-treatment ALC were 1.40 × 109, 0.23 × 109 and 0.28 × 109/L, respectively. Regression analysis confirmed that the primary tumor location, number of fractions and spleen V5 were significant predictors of Min ALC during radiation therapy. Changes in ALC (ΔALC) were identified as an independent predictor of both OS and DFS. Spleen V5 is an independent predictor for Min ALC, and the maximum dose of the spleen is associated with an increased risk of severe lymphopenia. Therefore, these doses should be restricted in clinical practice. Additionally, ΔALC can serve as a prognostic indicator for adjuvant radiotherapy in gastric cancer.

The injuries of spleen and intestinal immune system induced by 2-Gy 60Co γ-ray whole-body irradiation.

PURPOSE: The aim of the present study was to investigate the injuries of spleen and intestinal immune system induced by 2 Gy 60Co γ ray in mice. MATERIALS AND METHODS: A total of 120 Balb/c mice were randomly divided into two groups: blank control (Ctrl) and model (IR). The IR mice were exposed to a single dose of total body irradiation (2 Gy, dose rate: 1 Gy/min) and sacrificed on 1st, 3rd, 7th, 14th and 21st day after irradiation. The indicators including general observations and body weight, the changes in peripheral hemogram, spleen index, histopathology examination and lymphocyte subsets of spleen. As well as the count and subsets of lymphocyte in gut-associated lymphoid tissue. RESULTS: Compared with the Ctrl group, the body weight, spleen index, peripheral blood cell and splenocyte amounts, intraepithelial lymphocytes number decreased significantly after exposure, accompanied by a notable decreased count of lymphocytes in Peyer's patch and mesenteric lymph nodes. Moreover, ionizing radiation also broke the balance of CD4+/CD8+ and increased the Treg proportion in spleen, which then triggered immune imbalance and immunosuppression. In general, the spleen injuries occurred on 1st day after exposure, worse on 3rd day, and were relieved on 7th day. The intestinal immune injuries were observed on 1st day, and attenuated on 3rd day. On 21st day after exposure, the spleen volume and index have returned to normal, except for the distribution of lymphocyte subpopulations. Furthermore, all indicators of gut-associated lymphoid tissue, except for mesenteric lymph nodes lymphocyte count, had returned to normal levels on 21st day. CONCLUSION: In conclusion, our data showed the injuries of spleen and intestinal immune system induced by 2 Gy 60Co γ ray whole-body irradiation. These findings may provide the bases for further radiation protection in the immunity.

Preventive effect of sanguinarine on intestinal injury in mice exposed to whole abdominal irradiation.

Intestinal injury is one of the major side effects that are induced by medical radiation exposure, and has limited effective therapies. In this study, we investigated the beneficial effects of sanguinarine (SAN) on intestinal injury induced by ionizing radiation (IR) both in vitro and in vivo. Mice were exposed to whole abdominal irradiation (WAI) to mimic clinical scenarios. SAN was injected intraperitoneally to mitigate IR-induced injury. Histological examination was performed to assess the tissue injuries of the spleen and small intestine. A small intestinal epithelial cell line-6 (IEC-6) was analyzed for its viability and apoptosis in vitro under different treatments. Inflammation-related pathways and serum inflammatory cytokines were detected via Western blot analysis and ELISA, respectively. High-throughput sequencing was used to characterize the gut microbiota profile. High-performance liquid chromatography was performed to assess short-chain fatty acid contents in the colon. In vitro, SAN pretreatment protected cell viability and reduced apoptosis in IEC-6 cells. In vivo, SAN pretreatment protected immune organs, alleviated intestinal injury, and promoted intestinal recovery. SAN also reduced the levels of inflammatory cytokines, suppressed high mobility group box 1 (HMGB1)/ Toll-like receptor 4 (TLR4) pathway activation, and modulated gut microbiota composition. Our findings demonstrate that the beneficial properties of SAN alleviated intestinal radiation injury. Thus, SAN represents a therapeutic option for protecting against IR-induced intestinal injury in preclinical settings.

Yiqi Jiedu herbal decoction attenuates the 2 Gy 60Co γ ray induced spleen injury by inhibiting apoptosis and modulating the immune balance.

ETHNOPHARMACOLOGICAL RELEVANCE: Irradiation-induced immunosuppression often occurs during radiotherapy in patients, which would increase the risk of opportunistic infections. Many Chinese herbal prescriptions or natural extracts have recently attracted increased radiation protection and therapy attention due to their low toxicity. AIM OF THE STUDY: The present study aimed to investigate the protective effects of Yiqi Jiedu (YQJD) decoction on spleen injury induced by 2 Gy 60Co γ ray in mice. MATERIALS AND METHODS: A total of 180 Balb/c mice were randomly divided into five groups: blank control (Ctrl), model (IR), positive drug (IRA), low-dose YQJD decoction (IRL), and high-dose YQJD decoction (IRH). After a ten-day intervention, mice were exposed to a single dose of total body irradiation (2 Gy) and sacrificed on the 1st, 3rd, and 7th day after irradiation. The indicators include general observations and body weight, changes in peripheral hemogram, index and histopathology examination of the spleen, distribution of lymphocyte subsets, cytokine levels, and apoptosis in the spleen. RESULTS: In comparison to the Ctrl group, the body weight, spleen index, peripheral blood cell, and splenocyte quantities decreased significantly after exposure, accompanied by a notable increase of apoptosis in spleen cells. Moreover, ionizing radiation also broke the balance of CD4+/CD8+, Th1/Th2, and Th17/Treg, triggering immune imbalance and immunosuppression. The above injuries occurred on the 1st day after exposure, worsened on the 3rd, and were relieved on the 7th day. However, the pretreatment of YQJD decoction increased the spleen index, improved the spleen structure, and inhibited radiation-induced apoptosis after exposure. Additionally, YQJD decoction has shown its ability to promote immunological balance recovery following exposure by regulating CD4+/CD8+, Th1/Th2, and Th17/Treg ratios, which may minimize the risk of infection. In addition, the high-dose of YQJD decoction showed a better protective effect than the low-dose group. CONCLUSION: The protective effects of YQJD decoction on 2 Gy 60Coγray induced spleen injury were confirmed in this study. This mechanism may be related to inhibiting apoptosis and modulating immune balance. This exploration might provide new insights into the use of Chinese herbs on radioprotection of the immune system.

Vertebral body and splenic irradiation are associated with lymphopenia in localized pancreatic cancer treated with stereotactic body radiation therapy.

OBJECTIVES: The purpose of this study was to determine if vertebral body and splenic dosimetry was associated with the development of lymphopenia in patients with borderline resectable (BRPC) and locally advanced pancreatic cancer (LAPC) treated with stereotactic body radiation therapy (SBRT). METHODS: Patients with BRPC/LAPC who were treated with SBRT and who had lymphocyte counts and radiation treatment plans available for review were included in the study. Vertebral body levels T11-L3 and the spleen were retrospectively contoured for each patient. Univariate (UVA) and multivariable analyses (MVA) were performed to identify associations between vertebral body and splenic dosimetric parameters with absolute lymphocyte count (ALC) and grade ≥ 2 lymphopenia. Receiver operator characteristic curves were generated to identify dose-volume thresholds in predicting grade ≥ 2 lymphopenia. RESULTS: A total of 132 patients were included in the study. On UVA and MVA, vertebral V15 (regression coefficient [ß]: - 0.026, 95% CI - 0.044 to - 0.009, p = 0.003), vertebral V2.5 (ß: - 0.011, 95% CI - 0.020 to - 0.002, p = 0.015), and log10PTV (ß: - 0.15, 95% CI - 0.30 to - 0.005, p = 0.042) were associated with post-SBRT ALC. On UVA and MVA, vertebral V15 (odds ratio [OR]: 3.98, 95% CI 1.09-14.51, p = 0.027), vertebral V2.5 (OR: 1.04, 95% CI 1.00-1.09, p = 0.032), and spleen V10 (OR: 1.05, 95% CI 1.09-1.95, p = 0.004) were associated with development of grade ≥ 2 lymphopenia. Development of grade ≥ 2 lymphopenia was more likely in patients with vertebral V15 ≥ 5.84% (65.5% vs 34.0%, p = 0.002), vertebral V2.5 ≥ 48.36% (48.9% vs 23.8%, p = 0.005), and spleen V10 ≥ 4.17% (56.2% vs 26.9%, p < 0.001). CONCLUSIONS: Increasing radiation dose to vertebral bodies and spleen were associated with the development of lymphopenia in BRPC/LAPC treated with SBRT. Optimization of vertebral body and splenic dosimetry may reduce the risk of developing lymphopenia and improve clinical outcomes in this population.

Dose and Dose Rate-Dependent Effects of Low-Dose Irradiation on Inflammatory Parameters in ApoE-Deficient and Wild Type Mice.

Anti-inflammatory low-dose therapy is well established, whereas the immunomodulatory impact of doses below 0.1 Gy is much less clear. In this study, we investigated dose, dose rate and time-dependent effects in a dose range of 0.005 to 2 Gy on immune parameters after whole body irradiation (IR) using a pro-inflammatory (ApoE-/-) and a wild type mouse model. Long-term effects on spleen function (proliferation, monocyte expression) were analyzed 3 months, and short-term effects on immune plasma parameters (IL6, IL10, IL12p70, KC, MCP1, INFγ, TGFß, fibrinogen, sICAM, sVCAM, sE-selectin/CD62) were analyzed 1, 7 and 28 days after Co60 γ-irradiation (IR) at low dose rate (LDR, 0.001 Gy/day) and at high dose rate (HDR). In vitro measurements of murine monocyte (WEHI-274.1) adhesion and cytokine release (KC, MCP1, IL6, TGFß) after low-dose IR (150 kV X-ray unit) of murine endothelial cell (EC) lines (H5V, mlEND1, bEND3) supplement the data. RT-PCR revealed significant reduction of Ki67 and CD68 expression in the spleen of ApoE-/- mice after 0.025 to 2 Gy exposure at HDR, but only after 2 Gy at LDR. Plasma levels in wild type mice, showed non-linear time-dependent induction of proinflammatory cytokines and reduction of TGFß at doses as low as 0.005 Gy at both dose rates, whereas sICAM and fibrinogen levels changed in a dose rate-specific manner. In ApoE-/- mice, levels of sICAM increased and fibrinogen decreased at both dose rates, whereas TGFß increased mainly at HDR. Non-irradiated plasma samples revealed significant age-related enhancement of cytokines and adhesion molecules except for sICAM. In vitro data indicate that endothelial cells may contribute to systemic IR effects and confirm changes of adhesion properties suggested by altered sICAM plasma levels. The differential immunomodulatory effects shown here provide insights in inflammatory changes occurring at doses far below standard anti-inflammatory therapy and are of particular importance after diagnostic and chronic environmental exposures.

Tissue Sampling and Homogenization in the Sub-Microliter Scale with a Nanosecond Infrared Laser (NIRL) for Mass Spectrometric Proteomics.

It was recently shown that ultrashort pulse infrared (IR) lasers, operating at the wavelength of the OH vibration stretching band of water, are highly efficient for sampling and homogenizing biological tissue. In this study we utilized a tunable nanosecond infrared laser (NIRL) for tissue sampling and homogenization with subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis for mass spectrometric proteomics. For the first time, laser sampling was performed with murine spleen and colon tissue. An ablation volume of 1.1 × 1.1 × 0.4 mm³ (approximately 0.5 µL) was determined with optical coherence tomography (OCT). The results of bottom-up proteomics revealed proteins with significant abundance differences for both tissue types, which are in accordance with the corresponding data of the Human Protein Atlas. The results demonstrate that tissue sampling and homogenization of small tissue volumes less than 1 µL for subsequent mass spectrometric proteomics is feasible with a NIRL.

Glycyrrhizin ameliorating sterile inflammation induced by low-dose radiation exposure.

Glycyrrhizin (GL) is a direct inhibitor of HMGB1 which acts as an alarmin when excreted into the extracellular space. High-dose radiation in radiotherapy induces collateral damage to the normal tissue, which can be mitigated by GL inhibiting HMGB1. The purpose of this study was to assess changes in HMGB1 and pro-inflammatory cytokines and to evaluate the protective effect of GL after low-dose radiation exposure. BALB/c mice were irradiated with 0.1 Gy (n = 10) and 1 Gy (n = 10) with GL being administered to half of the mice (n = 5, respectively) before irradiation. Blood and spleen samples were harvested and assessed for oxidative stress, HMGB1, pro-inflammatory cytokines, and cell viability. HMGB1 and pro-inflammatory cytokines increased and cell viability decreased after irradiation in a dose-dependent manner. Oxidative stress also increased after irradiation, but did not differ between 0.1 Gy and 1 Gy. With the pretreatment of GL, oxidative stress, HMGB1, and all of the pro-inflammatory cytokines decreased while cell viability was preserved. Our findings indicate that even low-dose radiation can induce sterile inflammation by increasing serum HMGB1 and pro-inflammatory cytokines and that GL can ameliorate the sterile inflammatory process by inhibiting HMGB1 to preserve cell viability.

Roles of Simvastatin and Sildenafil in Modulation of Cranial Irradiation-Induced Bystander Multiple Organs Injury in Rats.

In radiobiology and radiation oncology fields, the observation of a phenomenon called radiation-induced bystander effect (RIBE) has introduced the prospect of remotely located tissues' affection. This phenomenon has been broadly developed to involve the concept of RIBE, which are relevant to the radiation-induced response of a distant tissue other than the irradiated one. The current study aimed at investigating each of the RIBE of cranial irradiation on oxidative and inflammatory status in different organs such as liver, kidney, heart, lung, and spleen. Being a vital target of the cholinergic anti-inflammatory response to an inflammatory stimulus, the splenic α-7-nicotinic acetylcholine receptor (α-7nAchR) was evaluated and the hepatic contents of thioredoxin, peroxisome proliferator-activated receptor-alpha and paraoxinase-1 (Trx/PPAR-α/PON) were also assessed as indicators for the liver oxidative stress and inflammatory responses. Being reported to act as antioxidant and anti-inflammatory agents, simvastatin (SV) and/or sildenafil (SD) were investigated for their effects against RIBE on these organs. These objectives were achieved via the biochemical assessments and the histopathological tissues examinations. Five experimental groups, one sham irradiated and four irradiated groups, were exposed to cranial irradiation at dose level of 25 Gy using an experimental irradiator with a Cobalt (Co60) source, RIBE, RIBE + SV (20 mg.(kg.bw)-1 day-1), RIBE + SD (75 mg.(kg.bw)-1 day-1), and RIBE + SV + SD. Cranial irradiation induced structural, biochemical, and functional dys-regulations in non-targeted organs. RIBE-induced organs' injuries have been significantly corrected by the administration of SV and/or SD. Our results suggest the possibility of a potentiated interaction between SV and SD in the modulation of the RIBE associated with head and neck radiotherapy.

Transcriptomic Response in the Spleen after Whole-Body Low-Dose X-Ray Irradiation.

As the use of medical radiation procedures continues to rise, it is imperative to further our understanding of the effects of this exposure. The spleen is not known as a particularly radiosensitive organ, although its tolerance to radiation is not well understood. Low-dose radiation exposure has been implicated in beneficial responses, particularly in cell death and DNA damage repair. In this study, adult male rats received 2, 20, 200 mGy or 4 Gy whole-body X-ray irradiation and the transcriptional response in the spleen was analyzed at 0.5, 4 and 24 h postirradiation. We analyzed expression of genes involved in apoptosis, cell cycle progression and DNA damage repair. As expected, 4 Gy irradiated animals demonstrated elevated expression of genes related to apoptosis at 0.5, 4 and 24 h postirradiation in the spleen. These animals also showed upregulation of DNA damage repair genes at 24 h postirradiation. Interestingly, the spleens of 20 mGy irradiated animals showed reduced apoptosis and cell cycle arrest compared to the spleens of sham-irradiated animals. These results further reveal that the cellular response in the spleen to whole-body irradiation differs between low- and high-dose irradiation.

Case Report: Splenic Irradiation for the Treatment of Chronic Active Antibody-Mediated Rejection in Kidney Allograft Recipients With De Novo Donor-Specific Antibodies.

Chronic active antibody-mediated rejection (AMR) in renal transplantation is usually refractory to current conventional treatment with rituximab, plasmapheresis (PP), and intravenous immunoglobulins (IVIG). Splenic irradiation has been reported to be effective in the rescue of early severe acute AMR after kidney transplantation; however, its effect in chronic active AMR has not been reported to date. In order to reduce donor-specific antibody (DSA) and prevent the progression of chronic AMR, we used repetitive low-dose splenic irradiation, together with rituximab and PP/IVIG, in two living-related kidney transplant recipients with pathologically diagnosed chronic active AMR and the presence of long-term class II-de novo DSA. DSA monitoring and repeated renal biopsy revealed significantly reduced DSA levels as well as alleviated glomerulitis and peritubular capillaritis in both patients after treatment, and these therapies may have played a role in delaying the progression of chronic AMR. Although DSA levels in both patients eventually rebounded to some extent after treatment, serum creatinine increased slowly in one patient during the 16-month follow-up period and remained stable in the other during the 12-month follow-up period. Given the poor efficacy of conventional treatment at present, splenic irradiation may still be one of the treatment options for chronic active AMR.

Senescence-associated secretory phenotype and activation of NF-κB in splenocytes of old mice exposed to irradiation at a young age.

DNA damage-induced cellular senescence is involved in aging. We reported previously that p53+/- mice subjected to irradiation at a young age exhibited an increased number of splenic lymphocytes in the S and G2/M phases. However, the detailed nature of splenic disorders in these mice is not fully understood. In this study, we investigated the effects on molecules in splenocytes, especially on senescence factors after early exposure of mice to radiation. Mice, 8- (young) or 17-, 30-, and 41-week-old (old) p53+/- were subjected to 3-Gy whole-body irradiation. Splenocytes were prepared at 56 weeks of age. Immunoblot showed that irradiation at 8 weeks enhanced the expression and phosphorylation of p53, cyclin-dependent kinase 2, cell division cycle 6, and the MDM2 proto-oncogene in splenocytes. However, these molecules were not affected by irradiation at 17, 30, and 41 weeks of age. Similarly, irradiation at 8, but not 17, 30, or 41 weeks, induced phosphorylation of IKKα, NF-κB inhibitor alpha, and p65. Electrophoretic mobility shift assay demonstrated that active forms of NF-κB were increased. In addition, enzyme-linked immunosorbent assay showed that lipopolysaccharide-induced IL-6 production was enhanced in splenocytes of mice irradiated at 8 weeks. ATP levels were increased in splenocytes of mice irradiated at 8, but not 17, 30, or 41 weeks. CDK2 expression and p65 phosphorylation were induced in CD45R/B220+ cells from irradiated mice. Overall, irradiation induced a NF-κB-related immune response in the spleen with an increase in senescence marker proteins, such as CDKs and IL-6, which are known to be typical senescence-associated secretory phenotype factors related to stresses, such as DNA damage.

A derivative of vitamin B3 applied several days after exposure reduces lethality of severely irradiated mice.

Most, if not all, of the hitherto tested substances exert more or less pronounced pro-survival effects when applied before or immediately after the exposure to high doses of ionizing radiation. In the present study we demonstrate for the first time that 1-methyl nicotinamide (MNA), a derivative of vitamin B3, significantly (1.6 to 1.9 times) prolonged survival of BALB/c mice irradiated at LD30/30 (6.5 Gy), LD50/30 (7.0 Gy) or LD80/30 (7.5 Gy) of γ-rays when the MNA administration started as late as 7 days post irradiation. A slightly less efficient and only after the highest dose (7.5 Gy) of γ-rays was another vitamin B3 derivative, 1-methyl-3-acetylpyridine (1,3-MAP) (1.4-fold prolonged survival). These pro-survival effects did not seem to be mediated by stimulation of haematopoiesis, but might be related to anti-inflammatory and/or anti-thrombotic properties of the vitamin B3 derivatives. Our results show that MNA may represent a prototype of a radioremedial agent capable of mitigating the severity and/or progression of radiation-induced injuries when applied several hours or days after exposure to high doses of ionizing radiation.

Synergistic Effects of Chronic Restraint-Induced Stress and Low-Dose 56Fe-particle Irradiation on Induction of Chromosomal Aberrations in Trp53-Heterozygous Mice.

Astronauts can develop psychological stress (PS) during space flights due to the enclosed environment, microgravity, altered light-dark cycles, and risks of equipment failure or fatal mishaps. At the same time, they are exposed to cosmic rays including high atomic number and energy (HZE) particles such as iron-56 (Fe) ions. Psychological stress or radiation exposure can cause detrimental effects in humans. An earlier published pioneering study showed that chronic restraint-induced psychological stress (CRIPS) could attenuate Trp53 functions and increase carcinogenesis induced by low-linear energy transfer (LET) γ rays in Trp53-heterozygous (Trp53+/-) mice. To elucidate possible modification effects from CRIPS on high-LET HZE particle-induced health consequences, Trp53+/- mice were received both CRIPS and accelerated Fe ion irradiation. Six-week-old Trp53+/- C57BL/6N male mice were restrained 6 h per day for 28 consecutive days. On day 8, they received total-body Fe-particle irradiation (Fe-TBI, 0.1 or 2 Gy). Metaphase chromosome spreads prepared from splenocytes at the end of the 28-day restraint regimen were painted with the fluorescence in situ hybridization (FISH) probes for chromosomes 1 (green), 2 (red) and 3 (yellow). Induction of psychological stress in our experimental model was confirmed by increase in urinary corticosterone level on day 7 of restraint regimen. Regardless of Fe-TBI, CRIPS reduced splenocyte number per spleen at the end of the 28-day restraint regimen. At 2 Gy, Fe-TBI alone induced many aberrant chromosomes and no modifying effect was detected from CRIPS on induction of aberrant chromosomes. Notably, neither Fe-TBI at 0.1 Gy nor CRIPS alone induced any increase in the frequency of aberrant chromosomes, while simultaneous exposure resulted in a significant increase in the frequency of chromosomal exchanges. These findings clearly showed that CRIPS could enhance the frequency of chromosomal exchanges induced by Fe-TBI at a low dose of 0.1 Gy.