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Calorie restriction (CR) suppresses not only spontaneous but also chemical- and radiation-induced carcinogenesis. Our previous study revealed that the cancer-preventive effect of CR is tissue dependent and that CR does not effectively prevent the development of thymic lymphoma (TL). We investigated the association between CR and the genomic alterations of resulting TLs to clarify the underlying resistance mechanism. TLs were obtained from previous and new experiments, in which B6C3F1 mice were exposed to radiation at 1 week of age and fed with a CR or standard (non-CR) diet from 7 weeks throughout their lifetimes. All available TLs were used for analysis of genomic DNA. In contrast to the TLs of the non-CR group, those of the CR group displayed suppression of copy-neutral loss of heterozygosity (LOH) involving relevant tumor suppressor genes (Cdkn2a, Ikzf1, Trp53, Pten), an event regarded as cell division-associated. However, CR did not affect interstitial deletions of those genes, which were observed in both groups. In addition, CR affected the mechanism of Ikzf1 inactivation in TLs: the non-CR group exhibited copy-neutral LOH with duplicated inactive alleles, whereas the CR group showed expression of dominant-negative isoforms accompanying a point mutation or an intragenic deletion. These results suggest that, even though CR reduces cell division-related genomic rearrangements by suppressing cell proliferation, tumors arise via diverse carcinogenic pathways including inactivation of tumor suppressors via interstitial deletions and other mutations. These findings provide a molecular basis for improved prevention strategies that overcome the CR resistance of lymphomagenesis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Nakayama et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The gastrin-releasing peptide receptor (GRPR) is a promising molecular target for imaging and therapy of prostate cancer using bombesin peptides that bind to the receptor with high affinity. Targeted copper theranostics (TCTs) using copper radionuclides, 64 Cu for imaging and 67 Cu for therapy, offer significant advantages in the development of next-generation theranostics. [ 64 Cu]Cu-SAR-BBN is in clinical development for PET imaging of GRPR-expressing cancers. This study explores the therapeutic efficacy of [ 67 Cu]Cu-SAR-BBN in a pre-clinical mouse model. The peptide was radiolabeled with 67 Cu, and specific binding of the radiolabeled peptide towards GRPR-positive PC-3 prostate cancer cells was confirmed with 52.2 ± 1.4% total bound compared to 5.8 ± 0.1% with blocking. A therapy study with [ 67 Cu]Cu-SAR-BBN was conducted in mice bearing PC-3 tumors by injecting 24 MBq doses a total of six times. Tumor growth was inhibited by 93.3% compared to the control group on day 19, and median survival increased from 34.5 days for the control group to greater than 54 days for the treatment group. The ease and stability of the radiochemistry, favorable biodistribution, and the positive tumor inhibition demonstrate the suitability of this copper-based theranostic agent for clinical assessment in the treatment of cancers expressing GRPR.

The risk of radiation-induced carcinogenesis depends on age at exposure. We previously reported principal causative genes in lymphomas arising after infant or adult exposure to 4-fractionated irradiation as Pten or Ikzf1 , respectively, suggesting that cells with mutation in these genes might be the origin of lymphomas arising after irradiation depending on age at exposure. Here, we clarified the age-dependent differences in thymus-cell dynamics in mice during the initial post-irradiation period. The thymocyte number initially decreased, followed by two regeneration phases. During the first regeneration, the proportion of phosphorylated-AKT-positive (p-AKT + ) cells in cell-cycle phases S+G2/M of immature CD4 - CD8 - and CD4 + CD8 + thymocytes and in phases G0/G1 of mature CD4 + CD8 - and CD4 - CD8 + thymocytes was significantly greater in irradiated infants than in irradiated adults. During the second regeneration, the proportion of p-AKT + thymocytes in phases G0/G1 increased in each of the three populations other than CD4 - CD8 - thymocytes more so than during the first regeneration. Finally, PI3K-AKT-mTOR signaling in infants contributed, at least in part, to biphasic thymic regeneration through the modification of cell proliferation and survival after irradiation, which may be associated with the risk of Pten mutation-associated thymic lymphoma.

Radionuclide therapy targeting prostate-specific membrane antigen (PSMA) is promising for prostate cancer. We previously reported a ligand, 64 Cu-CuSarbisPSMA, featuring 2 lysine-ureido-glutamate groups. Here, we report the therapeutic potential of 67 Cu-CuSarbisPSMA. Methods: Growth of PSMA-positive xenografts was evaluated after treatment with 67 Cu-CuSarbisPSMA or 177 Lu-LuPSMA imaging and therapy (I&T). Results: At 13 d after injection, tumor growth was similarly inhibited by the 2 tracers in a dose-dependent manner. Survival was comparable after single (30 MBq) or fractionated (2 × 15 MBq, 2 wk apart) administrations. Conclusion: 67 Cu-CuSarbisPSMA is efficacious in a PSMA-expressing model of prostate cancer., (© 2021 by the Society of Nuclear Medicine and Molecular Imaging.)

Background: Muscle wasting (Sarcopenia) is associated with poor outcomes in cancer patients. Early identification of sarcopenia can facilitate nutritional and exercise intervention. Cross-sectional skeletal muscle (SM) area at the third lumbar vertebra (L3) slice of a computed tomography (CT) image is increasingly used to assess body composition and calculate SM index (SMI), a validated surrogate marker for sarcopenia in cancer. Manual segmentation of SM requires multiple steps, which limits use in routine clinical practice. This project aims to develop an automatic method to segment L3 muscle in CT scans., Methods: Attenuation correction CTs from full body PET-CT scans from patients enrolled in two prospective trials were used. The training set consisted of 66 non-small cell lung cancer (NSCLC) patients who underwent curative intent radiotherapy. An additional 42 NSCLC patients prescribed curative intent chemo-radiotherapy from a second trial were used for testing. Each patient had multiple CT scans taken at different time points prior to and post- treatment (147 CTs in the training and validation set and 116 CTs in the independent testing set). Skeletal muscle at L3 vertebra was manually segmented by two observers, according to the Alberta protocol to serve as ground truth labels. This included 40 images segmented by both observers to measure inter-observer variation. An ensemble of 2.5D fully convolutional neural networks (U-Nets) was used to perform the segmentation. The final layer of U-Net produced the binary classification of the pixels into muscle and non-muscle area. The model performance was calculated using Dice score and absolute percentage error (APE) in skeletal muscle area between manual and automated contours., Results: We trained five 2.5D U-Nets using 5-fold cross validation and used them to predict the contours in the testing set. The model achieved a mean Dice score of 0.92 and an APE of 3.1% on the independent testing set. This was similar to inter-observer variation of 0.96 and 2.9% for mean Dice and APE respectively. We further quantified the performance of sarcopenia classification using computer generated skeletal muscle area. To meet a clinical diagnosis of sarcopenia based on Alberta protocol the model achieved a sensitivity of 84% and a specificity of 95%., Conclusions: This work demonstrates an automated method for accurate and reproducible segmentation of skeletal muscle area at L3. This is an efficient tool for large scale or routine computation of skeletal muscle area in cancer patients which may have applications on low quality CTs acquired as part of PET/CT studies for staging and surveillance of patients with cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Amarasinghe, Lopes, Beraldo, Kiss, Bucknell, Everitt, Jackson, Litchfield, Denehy, Blyth, Siva, MacManus, Ball, Li and Hardcastle.)

Radiation exposure during the peri-pubertal period is a proven risk factor for breast cancer, whereas parity is an established protective factor. The present study investigated whether parity imposes differential protective effects against radiation-induced rat mammary carcinoma depending on the age at exposure. Pre- and post-pubertal female rats, irradiated or left unirradiated, were mated and allowed to nurse until weaning or left unmated. Appearance of mammary tumors was monitored, and serum concentrations of estradiol and progesterone were measured following weaning. Carcinomas were evaluated by immunohistochemistry for estrogen receptor, progesterone receptor, and the cell proliferation marker Ki-67. Parity reduced the risk of carcinoma in unirradiated and pre-pubertally irradiated rats but not post-pubertally irradiated rats. Although radiation exposure increased serum progesterone level, parity after pre-pubertal exposure significantly decreased the elevated progesterone to a normal level, reflecting a protective effect. Moreover, parity significantly decreased the proportion of hormone receptor-positive carcinomas after pre-pubertal exposure. Parity was also related to the observed positive association between progesterone receptor and Ki-67 indices in cancer tissue, implying progesterone receptor-dependent cell proliferation. Thus, parity protects against radiation-induced rat mammary carcinogenesis depending on the age at exposure; the mechanisms may involve changes in hormone levels and cancer tissue.

Whilst the near instantaneous physical interaction of radiation energy with living cells leaves little opportunity for inter-individual variation in the initial yield of DNA damage, all the downstream processes in how damage is recognized, repaired or resolved and therefore the ultimate fate of cells can vary across the population. In the clinic, this variability is observed most readily as rare extreme sensitivity to radiotherapy with acute and late tissue toxic reactions. Though some radiosensitivity can be anticipated in individuals with known genetic predispositions manifest through recognizable phenotypes and clinical presentations, others exhibit unexpected radiosensitivity which nevertheless has an underlying genetic cause. Currently, functional assays for cellular radiosensitivity represent a strategy to identify patients with potential radiosensitivity before radiotherapy begins, without needing to discover or evaluate the impact of the precise genetic determinants. Yet, some of the genes responsible for extreme radiosensitivity would also be expected to confer susceptibility to radiation-induced cancer, which can be considered another late adverse event associated with radiotherapy. Here, the utility of functional assays of radiosensitivity for identifying individuals susceptible to radiotherapy-induced second cancer is discussed, considering both the common mechanisms and important differences between stochastic radiation carcinogenesis and the range of deterministic acute and late toxic effects of radiotherapy., Competing Interests: The authors declare no conflict of interest.

The PI3K/AKT pathway is one of the most important signaling networks in human breast cancer, and since it was potentially implicated in our preliminary investigations of radiation-induced rat mammary carcinomas, our aim here was to verify its role. We included mammary carcinomas induced by the chemical carcinogen 1-methyl-1-nitrosourea to determine whether any changes were radiation-specific. Most carcinomas from both groups showed activation of the PI3K/AKT pathway, but phosphorylation of AKT1 was often heterogeneous and only present in a minority of carcinoma cells. The negative pathway regulator Inpp4b was significantly downregulated in both groups, compared with in normal mammary tissue, and radiation-induced carcinomas also showed a significant decrease in Pten expression, while the chemically induced carcinomas showed a decrease in Pik3r1 and Pdk1. Significant upregulation of the positive regulators Erbb2 and Pik3ca was observed only in chemically induced carcinomas. However, no genes showed clear correlations with AKT phosphorylation levels, except in individual carcinomas. Only rare carcinomas showed mutations in PI3K/AKT pathway genes, yet these carcinomas did not exhibit stronger AKT phosphorylation. Thus, while AKT phosphorylation is a common feature of rat mammary carcinomas induced by radiation or a canonical chemical carcinogen, the mutation of key genes in the pathways or permanent changes to gene expression of particular signaling proteins do not explain the pathway activation in the advanced cancers. Although AKT signaling likely facilitates cancer development and growth in rat mammary carcinomas, it is unlikely that permanent disruption of the PI3K/AKT pathway genes is a major causal event in radiation carcinogenesis., (© The Author 2016. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.)

Several lines of evidence indicate one's age at exposure to radiation strongly modifies the risk of radiation-induced breast cancer. We previously reported that rat mammary carcinomas induced by pre- and post-pubertal irradiation have distinct gene expression patterns, but the changes underlying these differences have not yet been characterized. The aim of this investigation was to see if differences in CpG DNA methylation were responsible for the differences in gene expression between age at exposure groups observed in our previous study. DNA was obtained from the mammary carcinomas arising in female Sprague-Dawley rats that were either untreated or irradiated (γ-rays, 2 Gy) during the pre- or post-pubertal period (3 or 7 weeks old). The DNA methylation was analyzed using CpG island microarrays and the results compared to the gene expression data from the original study. Global DNA hypomethylation in tumors was accompanied by gene-specific hypermethylation, and occasionally, by unique tumor-specific patterns. We identified methylation-regulated gene expression candidates that distinguished the pre- and post-pubertal irradiation tumors, but these represented only 2 percent of the differentially expressed genes, suggesting that methylation is not a major or primary mechanism underlying the phenotypes. Functional analysis revealed that the candidate methylation-regulated genes were enriched for stem cell differentiation roles, which may be important in mammary cancer development and worth further investigation. However, the heterogeneity of human breast cancer means that the interpretation of molecular and phenotypic differences should be cautious, and take into account the co-variates such as hormone receptor status and cell-of-origin that may influence the associations., Competing Interests: The authors have declared that no competing interests exist.

The risk of radiation-induced cancer adds to anxiety in low-dose exposed populations. Safe and effective lifestyle changes which can help mitigate excess cancer risk might provide exposed individuals the opportunity to pro-actively reduce their cancer risk, and improve mental health and well-being. Here, we applied a mathematical multi-stage carcinogenesis model to the mouse lifespan data using adult-onset caloric restriction following irradiation in early life. We re-evaluated autopsy records with a veterinary pathologist to determine which tumors were the probable causes of death in order to calculate age-specific mortality. The model revealed that in both irradiated and unirradiated mice, caloric restriction reduced the age-specific mortality of all solid tumors and hepatocellular carcinomas across most of the lifespan, with the mortality rate dependent more on age owing to an increase in the number of predicted rate-limiting steps. Conversely, irradiation did not significantly alter the number of steps, but did increase the overall transition rate between the steps. We show that the extent of the protective effect of caloric restriction is independent of the induction of cancer from radiation exposure, and discuss future avenues of research to explore the utility of caloric restriction as an example of a potential post-irradiation mitigation strategy.

Monitoring mice exposed to carbon ion radiotherapy provides an indirect method to evaluate the potential for second cancer induction in normal tissues outside the radiotherapy target volume, since such estimates are not yet possible from historical patient data. Here, male and female B6C3F1 mice were given single or fractionated whole-body exposure(s) to a monoenergetic carbon ion radiotherapy beam at the Heavy Ion Medical Accelerator in Chiba, Japan, matching the radiation quality delivered to the normal tissue ahead of the tumour volume (average linear energy transfer = 13 keV x μm(-1)) during patient radiotherapy protocols. The mice were monitored for the remainder of their lifespan, and a large number of T cell lymphomas that arose in these mice were analysed alongside those arising following an equivalent dose of 137Cs gamma ray-irradiation. Using genome-wide DNA copy number analysis to identify genomic loci involved in radiation-induced lymphomagenesis and subsequent detailed analysis of Notch1, Ikzf1, Pten, Trp53 and Bcl11b genes, we compared the genetic profile of the carbon ion- and gamma ray-induced tumours. The canonical set of genes previously associated with radiation-induced T cell lymphoma was identified in both radiation groups. While the pattern of disruption of the various pathways was somewhat different between the radiation types, most notably Pten mutation frequency and loss of heterozygosity flanking Bcl11b, the most striking finding was the observation of large interstitial deletions at various sites across the genome in carbon ion-induced tumours, which were only seen infrequently in the gamma ray-induced tumours analysed. If such large interstitial chromosomal deletions are a characteristic lesion of carbon ion irradiation, even when using the low linear energy transfer radiation to which normal tissues are exposed in radiotherapy patients, understanding the dose-response and tissue specificity of such DNA damage could prove key to assessing second cancer risk in carbon ion radiotherapy patients.

Genetic, physiological and environmental factors are implicated in colorectal carcinogenesis. Mutations in the mutL homolog 1 (MLH1) gene, one of the DNA mismatch repair genes, are a main cause of hereditary colon cancer syndromes such as Lynch syndrome. Long-term chronic inflammation is also a key risk factor, responsible for colitis-associated colorectal cancer; radiation exposure is also known to increase colorectal cancer risk. Here, we studied the effects of radiation exposure on inflammation-induced colon carcinogenesis in DNA mismatch repair-proficient and repair-deficient mice. Male and female Mlh1(-/-) and Mlh1(+/+) mice were irradiated with 2 Gy X-rays when aged 2 weeks or 7 weeks and/or were treated with 1% dextran sodium sulfate (DSS) in drinking water for 7 days at 10 weeks old to induce mild inflammatory colitis. No colon tumors developed after X-rays and/or DSS treatment in Mlh1(+/+) mice. Colon tumors developed after DSS treatment alone in Mlh1(-/-) mice, and exposure to radiation prior to DSS treatment increased the number of tumors. Histologically, colon tumors in the mice resembled the subtype of well-to-moderately differentiated adenocarcinomas with tumor-infiltrating lymphocytes of human Lynch syndrome. Immunohistochemistry revealed that expression of both p53 and β-catenin and loss of p21 and adenomatosis polyposis coli proteins were observed at the later stages of carcinogenesis, suggesting a course of molecular pathogenesis distinct from typical sporadic or colitis-associated colon cancer in humans. In conclusion, radiation exposure could further increase the risk of colorectal carcinogenesis induced by inflammation under the conditions of Mlh1 deficiency., (© 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.)

The low dose radioadaptive response has been shown to be protective against high doses of radiation as well as aging-induced genomic instability. We hypothesised that a single whole-body exposure of low dose radiation would induce a radioadaptive response thereby reducing or abrogating aging-related changes in repeat element DNA methylation in mice. Following sham or 10 mGy X-irradiation, serial peripheral blood sampling was performed and differences in Long Interspersed Nucleic Element 1 (L1), B1 and Intracisternal-A-Particle (IAP) repeat element methylation between samples were assessed using high resolution melt analysis of PCR amplicons. By 420 days post-irradiation, neither radiation- or aging-related changes in the methylation of peripheral blood, spleen or liver L1, B1 and IAP elements were observed. Analysis of the spleen and liver tissues of cohorts of untreated aging mice showed that the 17-19 month age group exhibited higher repeat element methylation than younger or older mice, with no overall decline in methylation detected with age. This is the first temporal analysis of the effect of low dose radiation on repeat element methylation in mouse peripheral blood and the first to examine the long term effect of this dose on repeat element methylation in a radiosensitive tissue (spleen) and a tissue fundamental to the aging process (liver). Our data indicate that the methylation of murine DNA repeat elements can fluctuate with age, but unlike human studies, do not demonstrate an overall aging-related decline. Furthermore, our results indicate that a low dose of ionising radiation does not induce detectable changes to murine repeat element DNA methylation in the tissues and at the time-points examined in this study. This radiation dose is relevant to human diagnostic radiation exposures and suggests that a dose of 10 mGy X-rays, unlike high dose radiation, does not cause significant short or long term changes to repeat element or global DNA methylation.

The radioprotective agent amifostine is a free radical scavenger that can protect cells from the damaging effects of ionising radiation when administered prior to radiation exposure. However, amifostine has also been shown to protect cells from chromosomal mutations when administered after radiation exposure. As apoptosis is a common mechanism by which cells with mutations are removed from the cell population, we investigated whether amifostine stimulates apoptosis when administered after radiation exposure. We chose to study a relatively low dose which is the maximum radiation dose for radiation emergency workers (0.25 Gy) and a high dose relevant to radiotherapy exposures (6 Gy). Mice were administered 400 mg/kg amifostine 30 min before, or 3 h after, whole-body irradiation with 0.25 or 6 Gy X-rays and apoptosis was analysed 3 or 7 h later in spleen and bone marrow. We observed a significant increase in radiation-induced apoptosis in the spleen of mice when amifostine was administered before or after 0.25 Gy X-rays. In contrast, when a high dose of radiation was used (6 Gy), amifostine caused a reduction in radiation-induced apoptosis 3 h post-irradiation in spleen and bone marrow similar to previously published studies. This is the first study to investigate the effect of amifostine on radiation-induced apoptosis at a relatively low radiation dose and the first to demonstrate that while amifostine can reduce apoptosis from high doses of radiation, it does not mediate the same effect in response to low-dose exposures. These results suggest that there may be a dose threshold at which amifostine protects from radiation-induced apoptosis and highlight the importance of examining a range of radiation doses and timepoints.

The objective of the current study was to determine the classification accuracy of serum S100B and apolipoprotein (apoA-I) for mild traumatic brain injury (mTBI) and abnormal initial head computed tomography (CT) scan, and to identify ethnic, racial, age, and sex variation in classification accuracy. We performed a prospective, multi-centered study of 787 patients with mTBI who presented to the emergency department within 6 h of injury and 467 controls who presented to the outpatient laboratory for routine blood work. Serum was analyzed for S100B and apoA-I. The outcomes were disease status (mTBI or control) and initial head CT scan. At cutoff values defined by 90% of controls, the specificity for mTBI using S100B (0.899 [95% confidence interval (CI): 0.78-0.92]) was similar to that using apoA-I (0.902 [0.87-0.93]), and the sensitivity using S100B (0.252 [0.22-0.28]) was similar to that using apoA-I (0.249 [0.22-0.28]). The area under the receiver operating characteristic curve (AUC) for the combination of S100B and apoA-I (0.738, 95% CI: 0.71, 0.77), however, was significantly higher than the AUC for S100B alone (0.709, 95% CI: 0.68, 0.74, p=0.001) and higher than the AUC for apoA-I alone (0.645, 95% CI: 0.61, 0.68, p<0.0001). The AUC for prediction of abnormal initial head CT scan using S100B was 0.694 (95%CI: 0.62, 0.77) and not significant for apoA-I. At a S100B cutoff of <0.060 μg/L, the sensitivity for abnormal head CT was 98%, and 22.9% of CT scans could have been avoided. There was significant age and race-related variation in the accuracy of S100B for the diagnosis of mTBI. The combined use of serum S100B and apoA-I maximizes classification accuracy for mTBI, but only S100B is needed to classify abnormal head CT scan. Because of significant subgroup variation in classification accuracy, age and race need to be considered when using S100B to classify subjects for mTBI.

We present here the first high resolution melt (HRM) assay to quantitatively analyze differences in murine DNA methylation levels utilizing CpG methylation of Long Interspersed Elements-1 (LINE1 or L1). By calculating the integral difference in melt temperature between samples and a methylated control, and biasing PCR primers for unmethylated CpGs, the assay demonstrates enhanced sensitivity to detect changes in methylation in a cell line treated with low doses of 5-aza-2'-deoxycytidine (5-aza). The L1 assay was confirmed to be a good marker of changes in DNA methylation of L1 elements at multiple regions across the genome when compared with total 5-methyl-cytosine content, measured by Liquid Chromatography-Mass Spectrometry (LC-MS). The assay design was also used to detect changes in methylation at other murine repeat elements (B1 and Intracisternal-A-particle Long-terminal Repeat elements). Pyrosequencing analysis revealed that L1 methylation changes were non-uniform across the CpGs within the L1-HRM target region, demonstrating that the L1 assay can detect small changes in CpG methylation among a large pool of heterogeneously methylated DNA templates. Application of the assay to various tissues from Balb/c and CBA mice, including previously unreported peripheral blood (PB), revealed a tissue hierarchy (from hypermethylated to hypomethylated) of PB > kidney > liver > prostate > spleen. CBA mice demonstrated overall greater methylation than Balb/c mice, and male mice demonstrated higher tissue methylation compared with female mice in both strains. Changes in DNA methylation have been reported to be an early and fundamental event in the pathogenesis of many human diseases, including cancer. Mouse studies designed to identify modulators of DNA methylation, the critical doses, relevant time points and the tissues affected are limited by the low throughput nature and exorbitant cost of many DNA methylation assays. The L1 assay provides a high throughput, inexpensive and sensitive screening tool for identifying and characterizing DNA methylation changes to L1 elements at multiple regions across the genome.

Serum S100B elevations accurately reflect blood-brain barrier (BBB) damage. Because S100B is also present in peripheral tissues, release of this protein may not be specific to central nervous system (CNS) injury. Ubiquitin C-terminal hydrolase 1 (UCHL1), and phosphorylated neurofilament heavy chain (pNF-H) are found exclusively in neurons, but their relationship to BBB dysfunction has not been determined. The objective of this study was to determine the accuracy of serum UCHL1 and pNF-H as measures of BBB integrity after traumatic brain injury (TBI), to and compare them to S100B. We performed a prospective study of 16 patients with moderate to severe TBI (Glasgow Coma Scale [GCS] score ≤12) and 6 patients with non-traumatic headache who had cerebrospinal fluid (CSF) collected by ventriculostomy or lumbar puncture (LP). Serum and CSF were collected at the time of LP for headache patients and at 12, 24, and 48 h after injury for TBI patients. BBB function was determined by calculating albumin quotients (Q(A)), where Q(A)=[albumin(CSF)]/[albumin(serum)]. S100B, UCHL1, and pNF-H were measured by enzyme-linked immunosorbent assay (ELISA). Pearson's correlation coefficient and area under the receiver operator characteristic (ROC) curve were used to determine relationships between serum markers and Q(A). At 12 hours after TBI, a significant relationship was found between Q(A) and serum UCHL1 concentrations (AUC=0.76; 95% CI 0.55,1.00), and between Q(A) and serum S100B concentrations (AUC=0.794; 95% CI 0.57,1.02). There was no significant relationship found between these markers and Q(A) at other time points, or between pNF-H and Q(A) at any time point. We conclude that serum concentrations of UCHL1 are associated with abnormal BBB status 12 h after moderate to severe TBI. This relationship is similar to that observed between serum S100B and Q(A,) despite the fact that S100B may be released from peripheral tissues after multi-trauma. We conclude that peripheral release of S100B after multi-trauma is probably negligible and that UCHL1 may have some utility to monitor BBB disruption following TBI.

The term radiation-induced bystander effect is used to describe radiation-induced biological changes that manifest in unirradiated cells remaining within an irradiated cell population. Despite their failure to fit into the framework of classical radiobiology, radiation-induced bystander effects have entered the mainstream and have become established in the radiobiology vocabulary as a bona fide radiation response. However, there is still no consensus on a precise definition of radiation-induced bystander effects, which currently encompasses a number of distinct signal-mediated effects. These effects are classified here into three classes: bystander effects, abscopal effects and cohort effects. In this review, the data have been evaluated to define, where possible, various features specific to radiation-induced bystander effects, including their timing, range, potency and dependence on dose, dose rate, radiation quality and cell type. The weight of evidence supporting these defining features is discussed in the context of bystander experimental systems that closely replicate realistic human exposure scenarios. Whether the manifestation of bystander effects in vivo is intrinsically limited to particular radiation exposure scenarios is considered. The conditions under which radiation-induced bystander effects are induced in vivo will ultimately determine their impact on radiation-induced carcinogenic risk.

To test whether bystander effects occur in vivo after low doses of radiation relevant to occupational and population exposure, we exposed mice to whole-body X-radiation doses (0.01 and 1 mGy) where only a proportion of cells would receive an electron track. We used a precise method to analyze the apoptosis frequency in situ in spleen tissue sections at 7 h and 1, 3 and 7 days after irradiation to determine whether an increase in apoptosis above that predicted by direct effects was observed. No significant changes in the apoptosis frequency at any time after low-dose irradiation were detected. Apoptosis was induced above endogenous levels by five- to sevenfold 7 h after 1000 mGy. Using these data, the expected increases in apoptosis 7 h after a dose of 1 mGy or 0.01 mGy were calculated based on the assumption that induction of apoptosis would decrease linearly with dose. The magnitude of potential bystander effects for apoptosis that could be detected above homeostatic levels after these low doses of radiation was determined. A substantial bystander effect for apoptosis (>50-fold above direct effects) would be required before such proposed effects would be identified using 10 animals/treatment group as studied here. These data demonstrate that amplification of apoptosis even due to a substantial bystander effect would fall within the homeostatic range.

The potential for irradiated cells to induce biological effects in their unirradiated neighbors (known as the bystander effect) has been observed repeatedly in vitro. However, whether bystander effects occur in vivo under the specific conditions relevant to low-dose radiation protection is still unclear. To test this, the fate of bystander cells in the mouse spleen was examined using an adoptive transfer method designed to replicate the rare, irradiated cells in an organ that might be expected after a low-dose-rate, low-LET radiation exposure. Splenic lymphocytes radiolabeled with low activities of (3)H-thymidine were introduced into the spleens of unirradiated recipient mice. In this study, the apoptotic and proliferative response of the neighboring bystander spleen cells was compared to the response of spleen cells in parallel control recipients that received sham-irradiated cells. Neither the local area surrounding lodged radiolabeled cells nor the spleen as a whole showed a change in apoptosis or proliferation either 1 or 3 days after adoptive transfer. Increasing the irradiated cell numbers, increasing the mean (3)H-thymidine activity per cell, or exposing cells ex vivo to an acute X-ray dose also had no effect. Possible reasons for the absence of a bystander effect in the spleen under these conditions are discussed.

The blood-brain barrier (BBB), which prevents the entry into the central nervous system (CNS) of most water-soluble molecules over 500 Da, is often disrupted after trauma. Post-traumatic BBB disruption may have important implications for prognosis and therapy. Assessment of BBB status is not routine in clinical practice because available techniques are invasive. The gold-standard measure, the cerebrospinal fluide (CSF)-serum albumin quotient (Q(A)), requires the measurement of albumin in CSF and serum collected contemporaneously. Accurate, less invasive techniques are necessary. The objective of this study was to evaluate the relationship between Q(A) and serum concentrations of monomeric transthyretin (TTR) or S100B. Nine subjects with severe traumatic brain injury (TBI; Glasgow Coma Scale [GCS] score < or =8) and 11 subjects with non-traumatic headache who had CSF collected by ventriculostomy or lumbar puncture (LP) were enrolled. Serum and CSF were collected at the time of LP for headache subjects and at 12, 24, and 48 h after ventriculostomy for TBI subjects. The Q(A) was calculated for all time points at which paired CSF and serum samples were available. Serum S100B and TTR levels were also measured. Pearson's correlation coefficient and area under the receiver operating characteristic (ROC) curve were used to determine the relationship between the serum proteins and QA. Seven TBI subjects had abnormal Q(A)'s indicating BBB dysfunction. The remaining TBI and control subjects had normal BBB function. No significant relationship between TTR and QA was found. A statistically significant linear correlation between serum S100B and Q(A) was present (r = 0.432, p = 0.02). ROC analysis demonstrated a significant relationship between Q(A) and serum S100B concentrations at 12 h after TBI (AUC = 0.800; SE 0.147, 95% CI 0.511-1.089). Using an S100B concentration cutoff of 0.027 ng=ml, specificity for abnormal Q(A) was 90% or higher at each time point. We conclude that serum S100B concentrations accurately indicate BBB dysfunction at 12 h after TBI.

Virgin, ovariectomized rats exposed to 2 wk of sequential estradiol (E(2)) and progesterone (P) followed by P withdrawal have increased hypothalamic oxytocin (OT) mRNA and peptide levels relative to sham-treated animals. This increase is prevented if P is sustained. In the central nervous system, P is metabolized to the neurosteroid allopregnanolone (3alpha-hydroxy-5alpha-pregnan-20-one), which exerts effects by acting as a positive allosteric modulator of GABA(A) receptor/Cl(-)-channel complexes. In the present study, ovariectomized rats that received sequential E(2) and P for 2 wk followed by P withdrawal were administered allopregnanolone at the time of P withdrawal. Hypothalamic and plasma allopregnanolone concentrations, serum E(2) and P concentrations, and hypothalamic OT mRNA levels were measured at death. Steroid-induced increases in OT mRNA were attenuated in animals treated with allopregnanolone at the time of P withdrawal. The results suggest that allopregnanolone plays an important modulatory role in steroid-mediated increases in hypothalamic OT.