Irradiated Lymphocytes Research Articles

A Phase-I Clinical Trial of Donor-Derived MIC Cell Infusion for the Induction of Donor-Specific Hyporesponsiveness after Living Donor Kidney Transplantation (TOL-1 study)

MIC cells are donor-derived monocytes that gain immunosuppressive properties after incubation with the proliferation inhibitor mitomycin C. PBMCs were harvested from living donors by leukapheresis and MIC cells were manufactured under GMP conditions. Kidney transplant recipients received either 1.5x10E6 MIC cells per kg body weight on day −2 (N=3, group A), or 1.5x10E8 MIC cells per kg body weight on day −2 (N=3, group B) or day −7 (N=4, group C) before living donor kidney transplantation. Patients received immunosuppressive therapy with CyA, EC-MPS and CS. Primary outcome measure was the frequency of adverse events (AE) on day 30. A total of 70 AEs including 4 severe AEs occurred in treated patients that were unrelated to MIC cell infusion. No positive cross match results, de novo donor specific antibodies, or rejection episodes but 2 infectious complications were recorded. Median serum creatinine on day 30 was 1.4 mg/dL with no significant proteinuria. In vitro, MIC cells were capable of inducing tolerogenic dendritic cells with low expression of costimulatory molecules CD80, CD86 and a 30% increase of immunosuppressive molecule CD103. During the observation phase beyond day 30 after surgery, serum creatinine remained stable (median 1.48 mg/dL on day 180) with no significant proteinuria (median 10 g/mol creatinine on day 180) and no rejection episodes. The patients from group C who received low-dose CyA and low-dose EC-MPS during the observation phase had no or only minimal reactivity against irradiated donor lymphocytes in mixed lymphocyte culture while reactivity against 3rd party lymphocytes was preserved. CD19+ B cells increased to a median of 300/μL until day 30 but decreased to a median of 35/μL on day 180. CD19+CD24highCD38high transitional Bregs increased from a median of 2% on day 30 to a median of 20% of the total CD19+ B cell pool on day 180. There was a strong increase in the plasma IL-10/TNFalpha ratio from a median of 0.05 before cell therapy to a median of 0.11 on day 180. In summary, MIC cell therapy represents a promising option for individualized immunosuppression after living donor kidney transplantation.

Evaluation of an Immunomodulator Drug as a Radioprotectant on Human Peripheral Blood Lymphocytes In Vitro

Background: IMOD™, a selenium enriched extract of the plants Tanacetum vulgare, Urtica dioica, and Rosa canina, has an excellent effect on oxidative stress. In this study, we investigated the radioprotective effects of this immunomodulatory drug on human peripheral blood lymphocytes.Methods: Peripheral blood samples obtained from venipuncture of the brachial vein were treated with IMOD™ (5, 10, 15, 20 μl) for 30 min and Cobalt 60 γ-rays (0.25, 0.5, 1, 2 Gy) as the test groups and cultured with the control. We used the micronuclei assay, cell death detection, and cell toxicity assay to analyze the treatment effects.Results: The frequency of micronuclei were 1.66 (0 Gy), 5.33 (0.25 Gy), 9.67 (0.5 Gy), 17.67 (1 Gy), and 23.67 (2 Gy) in the irradiated lymphocytes (P<0.001). The percentage of micronuclei frequency reduced to 20%, 26.83%, 37.68%, 16%, and 20.47% with IMOD™. Apoptosis and necrosis decreased significantly in the IMOD™ treated groups (P<0.05).Conclusion: IMOD™ may protect these cells against ionizing radiation.

THE IMPACT OF ASTAXANTHIN ON THE LEVEL OF DNA METHYLATION IN IRRADIATED IN VITRO HUMAN LYMPHOCYTES.

To investigate an effect of astaxanthin on global DNA methylation in human peripheral blood lympho-cytes exposed to γ-radiation in vitro. Samples of whole blood were diluted with RPMI-1640 medium in proportion 1 : 10 and incubated at 37 °Cfor 3 h. Samples were exposed to γ-ray (emitter IBL-237C, dose-rate 2.34 Gy/min) in dose 1.0 Gy. Astaxanthin wasadded into culture medium in final concentrations 20.0 μg/ml before irradiation. The analysis of the global DNAmethylation state was carried out using modified method of neutral single-cell gel electrophoresis (Comet assay)after digestion with the methylation-sensitive endonuclease HpaII. For every experiment the analysis of the frequency distribution of «comets» was carried out. The treatmentof non-irradiated and irradiated human peripheral blood lymphocytes with astaxanthin resulted to a statisticallysignificant (p < 0.05) shift of comet distribution with tendency to increasing level of DNA migration which indicatesincrease in the pool of cells with a reduced level of DNA methylation. It has been found that astaxanthin in concentration of 20.0 μg/ml reduced the levels of global DNAmethylation both in irradiated in vitro and native human lymphocytes. It suggests that astaxanthin has an effect onmechanisms of epigenetic regulation of gene expression.

Radioprotective and immunomodulatory effects of Mesua ferrea (Linn.) from Western Ghats of India., in irradiated Swiss albino mice and splenic lymphocytes

The present study reports the efficacy of Mesua ferrea (Linn.) extract in mitigating radiation induced toxicity along with immunomodulatory property. Swiss albino mice, splenic lymphocytes and plasmid pBR322 DNA were used to evaluate the radioprotective effect by exposing them to 6Gy EBR or 4 Gy gamma radiation. In this study, Mesua ferrea methanolic (MfM) or aqueous extracts (MfA) significantly protected pBR322 DNA against radiation induced strand breaks. Both the extracts significantly offered protection against radiation-induced apoptosis as indicated by propidium iodide staining and DNA ladder assay. MfM or MfA significantly scavenged radiation derived free radicals indicating their antioxidant potential. MfM or MfA were orally administered to Swiss albino mice at 250 or200 mg/kg body weight respectively for 7 days. The study showed significant increase in the levels of glutathione, and activities of endogenous antioxidant enzymes superoxide dismutase & catalase. Administration of MfA or MfM to mice significantly reduced electron beam radiation (EBR; 6Gy) induced increase in MDA levels. Immunomodulatory efficacy of MfM and MfA was evaluated using concanavalin-A (Con-A) induced proliferation of CFSE labelled splenic lymphocytes. Both the extracts significantly reduced proliferation in a dose-dependent manner. Further, MfM or MfA treatment prevented EBR induced histopathological changes in jejunum, spleen, liver and kidney. This demonstrates that the plant Mesua ferrea has promising antioxidant, radioprotective and immunomodulatory activity which may be attributed to the active constituents present in it.

Immunofluorescence Microscopy of γH2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks.

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas including genome integrity, genotoxicity, radiation biology, aging, cancer, and drug development. In response to DSB, the histone H2AX is phosphorylated at Serine 139 in a region of several megabase pairs forming discrete nuclear foci detectable by immunofluorescence microscopy. In addition, 53BP1 (p53 binding protein 1) is another important DSB-responsive protein promoting repair of DSB by nonhomologous end-joining while preventing homologous recombination. According to the specific functions of γH2AX and 53BP1, the combined analysis of γH2AX and 53BP1 by immunofluorescence microscopy may be a reasonable approach for a detailed analysis of DSB. This manuscript provides a step-by-step protocol supplemented with methodical notes for performing the technique. Specifically, the influence of the cell cycle on γH2AX foci patterns is demonstrated in normal fibroblasts of the cell line NHDF. Further, the value of the γH2AX foci as a biomarker is depicted in x-ray irradiated lymphocytes of a healthy individual. Finally, genetic instability is investigated in CD34+ cells of a patient with acute myeloid leukemia by immunofluorescence microscopy of γH2AX and 53BP1.

6,7,3′,4′-Tetrahydroxyisoflavone improves the survival of whole-body-irradiated mice via restoration of hematopoietic function

Purpose: 6,7,3′,4′-tetrahydroxyisoflavone (T3) is a novel chemically synthesized compound reported in our previous study. This study was designed to explore the radioprotective effect of T3, and if so, its potential mechanisms.Materials and methods: KunMing mice were exposed to various doses of γ irradiation (60 Co) after being treated with dimethyl sulfoxide (DMSO) or T3. Briefly, survival rate, dose reducing factor (DRF), body weight change (%), spleen index (SI) and thymus index (TI) of irradiated mice with or without different doses of T3 treatment were evaluated routinely. The hematopoietic function of bone marrow was emphatically investigated. In vitro experiments were performed to observe the protective effect of T3 on irradiated human lymphocyte AHH-1 cells by cell viability or flow cytometry (FCM) assays.Results: A single dose of subcutaneous administration of T3 significantly improved the survival rate, and enhanced the restoration of hematopoietic function in irradiated mice. T3 also decreased the apoptosis of irradiated AHH-1 cells in vitro.Conclusions: T3 protected mice against lethal γ irradiation-induced injury probably through the restoration of hematopoietic function. This implied that T3 could be further developed as a radioprotector.

Possible benefits of tomato juice consumption: a pilot study on irradiated human lymphocytes from healthy donors

BackgroundReactive oxygen species (ROS) mediate much of the DNA damage caused by ionizing radiation. Among carotenoids, lycopene and β-carotene, present in tomato juice, are known to be strong radical scavengers. The aim of the study was to investigate the effect of tomato juice intake on the levels of DNA damage and oxidative stress in human whole blood induced by in vitro exposure to X-rays.MethodsTen healthy adults were asked to drink 190 g of tomato juice, containing 17 mg lycopene and 0.25 mg β-carotene, per day for 3 weeks and then refrain from drinking it for 3 weeks. Peripheral whole blood samples were collected before and after the intake period of tomato juice and after the washout period. The blood samples were exposed in vitro to X-ray doses of 0, 0.1, 0.5, and 2 Gy. Cytogenetic damage was measured using the cytokinesis-block micronucleus (CBMN) assay and the dicentrics (DIC) assay. The level of oxidative stress was determined using serum 8-oxo-7, 8-dihydro-2-deoxyguanosine (8-oxo-dG) and plasma reactive oxygen metabolite-derived compounds (d-ROMs). The concentration of carotenoids in plasma was measured at the three time points.ResultsThe levels of 8-oxo-dG tended to decrease during the intake period and increase during the washout period. A non-significant inverse correlation was noted between the plasma concentration of lycopene plus β-carotene and the level of 8-oxo-dG (P = 0.064). The radiation-induced MN and DIC frequencies increased in a dose-dependent manner, and when compared at the same dose, the MN and DIC frequencies decreased during the intake period compared with those at baseline and then increased during the washout period. The results suggest that continuous tomato juice consumption non-significantly decreases extracellular 8-oxo-dG, d-ROMs, and MN. Tomato juice intake had minimal or no effect on radiation-induced 8-oxo-dG and d-ROMs. For most radiation doses, continuously tomato juice intake lowered the levels of MN and DIC.ConclusionTomato juice consumption may suppress human lymphocyte DNA damage caused by radiation, but further examination is required.Trial registration2014-001 and 2014-R06.

Prostate Cancer Patients with Late Radiation Toxicity Exhibit Reduced Expression of Genes Involved in DNA Double-Strand Break Repair and Homologous Recombination.

Severe late damage to normal tissue is a major limitation of cancer radiotherapy in prostate cancer patients. In a recent retrospective study, late radiation toxicity was found to relate to a decreased decay of γ-H2AX foci and reduced induction of DNA double-strand break repair genes. Here, we report evidence of prognostic utility in prostate cancer for γ-H2AX foci decay ratios and gene expression profiles derived from ex vivo-irradiated patient lymphocytes. Patients were followed ≥2 years after radiotherapy. Clinical characteristics were assembled, and toxicity was recorded using the Common Terminology Criteria (CTCAE) v4.0. No clinical factor was correlated with late radiation toxicity. The γ-H2AX foci decay ratio correlated negatively with toxicity grade, with a significant difference between grade ≥3 and grade 0 patients (P = 0.02). A threshold foci decay ratio, determined in our retrospective study, correctly classified 23 of 28 patients with grade ≥3 toxicity (sensitivity 82%) and 9 of 14 patients with grade 0 toxicity (specificity 64%). Induction of homologous recombination (HR) repair genes was reduced with increasing toxicity grade. The difference in fold induction of the HR gene set was most pronounced between grade 0 and grade ≥3 toxicity (P = 0.008). Notably, reduced responsiveness of HR repair genes to irradiation and inefficient double-strand break repair correlated with severe late radiation toxicity. Using a decay ratio classifier, we correctly classified 82% of patients with grade ≥3 toxicity, suggesting a prognostic biomarker for cancer patients with a genetically enhanced risk for late radiation toxicity to normal tissues after radiotherapy. Cancer Res; 77(6); 1485-91. ©2017 AACR.

Study the impact of astaxanthin on developing of genomic instability in human peripheral blood lymphocytes irradiated in vitro on G2 phase of cell cycle

To identify the possibility of modification by astaxanthin the level of genome damages induced by gamma quanta in the culture of human peripheral blood lymphocytes exposed in vitro on postsynthetic (G2) phase of the first mitotic cycle. Peripheral blood lymphocytes from four apparently healthy volunteers 35-51 years old were cultivated using modified micromethod. To obtain genomic damages in G2 phase of the first mitotic cycle the part of cultures was irradiated by γ quanta in dose 1.0 Gy through 46 hours of cultivation. Astaxanthin in final con centration 20 μg/ml was exposed to lymphocytes' cultures before the irradiation. Cytogenetic analysis the uniform ly stained slides of metaphase chromosomes was carried out to determine the frequencies of chromosome and chro matid types of aberrations. Using the method of individual cells electrophoresis (Comet assay) the relative level of DNA damages (Tail Moment index) and the frequency of apoptotic cells with high level of DNA fragmentation were evaluated. Mean group frequencies of chromosome aberrations after gamma irradiation of lymphocytes in vitro exceed ed those without radiation exposure and were 72.35 ± 1.17 and 2.46 ± 0.30 per 100 metaphases, respectively (p < 0.001), mainly due to chromatid type of aberrations (58.32 ± 1.29 per 100 metaphases). Adding of astaxanthin into culture medium before the irradiation did not result in changes as in the frequency of chromosomal damages (71.54 ± 1.34 per 100 metaphases) as in the spectrum of aberrations - also prevailed chromatid type of aberrations (58.47 ± 1.47 per 100 metaphases). The increase of Tail Moment index after radiation exposure (from 3.84 ± 0.36 to 12.06 ± 1.88, respectively, p < 0.001) and lack of significant impact of astaxanthin on this index in the irradiated lym phocytes (8.96 ± 2.39, p > 0.05) was established, ie astaxanthin didn't change the relative level of radiation induced DNA damages. Also apoptogenic effect of astaxanthin was not found: frequency of apoptotic cells were (2.25 ± 1.49) % in cultures of intact lymphocytes, (2.08 ± 1.54) % in irradiated cultures and (1.78 ± 1.25) % under joint action of gamma radiation and astaxanthin (p > 0.05). Noimpactofastaxanthinongenomicinstabilityinducedbygammairradiation invitroinculturesof human peripheral blood lymphocytes on postsynthetic (G2) phase of first mitotic cycle had been established.

Celecoxib mitigates genotoxicity induced by ionizing radiation in human blood lymphocytes.

Ionizing radiation causes DNA damage and chromosome abbreviations on normal cells. The radioprotective effect of celecoxib (CLX) was investigated against genotoxicity induced by ionizing radiation in cultured human blood lymphocytes. Peripheral blood samples were collected from human volunteers and were incubated at different concentrations at 1, 5, 10 and 50 μM of CLX for two hours. At each dose point, the whole blood was exposed in vitro to 150 cGy of X-ray, and then the lymphocytes were cultured with mitogenic stimulation to determine the micronucleus frequency in cytokinesis blocked binucleated lymphocytes. Incubation of the whole blood with CLX exhibited a significant decrease in the incidence of micronuclei in lymphocytes induced by ionizing radiation, as compared with similarly irradiated lymphocytes without CLX treatment. The maximum reduction on the frequency of micronuclei was observed at 50 μM of CLX (65% decrease). This data may have an important possible application for the protection of human lymphocytes from the genetic damage induced by ionizing irradiation in human exposed to radiation.

The radioprotective effect of metformin against cytotoxicity and genotoxicity induced by ionizing radiation in cultured human blood lymphocytes

Metformin is a widely prescribed drug used in the treatment of patients with type 2 diabetes. In this study, the radioprotective effect of metformin was investigated against cytotoxicity and genotoxicity induced by ionizing radiation (IR) in human peripheral blood lymphocytes. Human lymphocytes were treated with metformin at concentrations 10 and 50μM for 2h and irradiated with 6MV X-rays. The radiation antagonistic potential of metformin was assessed by MTT [3-(4,5-dimethyl-2-thiaozolyl)-2,5-diphenyl-2H tetrazolium bromide] assay, chromosomal aberration (CA) analysis, cytokinesis blocked micronucleus (CBMN) assay, and flow cytometry. Observations demonstrated a radiation-dose-dependent decrease in the percentage of cell viability after 24h. It was found that pretreatment with metformin (10 and 50μM) increased the percentage of cell viability. A highly significant dose modifying factor (DMF) 1.35 and 1.42 was observed for 10 and 50μM metformin, respectively. Metformin (10 and 50μM) pretreatment significantly decreased the frequency of dicentrics (DCs), acentric fragments (AFs), rings (RIs), micronuclei (MN), and nucleoplasmic bridges (NPBs) in irradiated human peripheral blood lymphocytes. Also, treatment with metformin (10 and 50μM) without irradiation did not increase the number of MN, NPBs, DCs, AFs, RIs, and did not show a cytostatic effect in the human peripheral blood lymphocytes. On the other hand, metformin treatment (10 and 50μM) 2h prior to irradiation significantly reduced X-radiation-induced apoptotic incidence in human lymphocytes. The present study demonstrates metformin to be an effective radioprotector against DNA damage and apoptosis induced by IR in human lymphocytes. These data have an important application for the protection of lymphocytes from the genetic damage and side-effects induced by radiotherapy in cancer patients.

Stable, Nonviral Expression of Mutated Tumor Neoantigen-specific T-cell Receptors Using the Sleeping Beauty Transposon/Transposase System.

Neoantigens unique to each patient's tumor can be recognized by autologous T cells through their T-cell receptor (TCR) but the low frequency and/or terminal differentiation of mutation-specific T cells in tumors can limit their utility as adoptive T-cell therapies. Transfer of TCR genes into younger T cells from peripheral blood with a high proliferative potential could obviate this problem. We generated a rapid, cost-effective strategy to genetically engineer cancer patient T cells with TCRs using the clinical Sleeping Beauty transposon/transposase system. Patient-specific TCRs reactive against HLA-A*0201-restriced neoantigens AHNAK(S2580F) or ERBB2(H473Y) or the HLA-DQB*0601-restricted neoantigen ERBB2IP(E805G) were assembled with murine constant chains and cloned into Sleeping Beauty transposons. Patient peripheral blood lymphocytes were coelectroporated with SB11 transposase and Sleeping Beauty transposon, and transposed T cells were enriched by sorting on murine TCRβ (mTCRβ) expression. Rapid expansion of mTCRβ(+) T cells with irradiated allogeneic peripheral blood lymphocytes feeders, OKT3, interleukin-2 (IL-2), IL-15, and IL-21 resulted in a preponderance of effector (CD27(-)CD45RA(-)) and less-differentiated (CD27(+)CD45RA(+)) T cells. Transposed T cells specifically mounted a polyfunctional response against cognate mutated neoantigens and tumor cell lines. Thus, Sleeping Beauty transposition of mutation-specific TCRs can facilitate the use of personalized T-cell therapy targeting unique neoantigens.

Towards Establishing Capacity for Biological Dosimetry at Ghana Atomic Energy Commission.

The aim of this study was not only to obtain basic technical prerequisites for the establishment of capacity of biological dosimetry at the Ghana Atomic Energy Commission (GAEC) but also to stimulate interest in biological dosimetry research in Ghana and Sub-Saharan Africa. Peripheral blood from four healthy donors was exposed to different doses (0–6 Gy) of gamma rays from a radiotherapy machine and lymphocytes were subsequently stimulated, cultured, and processed according to standard protocols for 48–50 h. Processed cells were analyzed for the frequencies of dicentric and centric ring chromosomes. Radiation dose delivered to the experimental model was verified using GafChromic® EBT films in parallel experiments. Basic technical prerequisites for the establishment of capacity of biological dosimetry in the GAEC have been realized and expertise in the dicentric chromosome assay consolidated. We successfully obtained preliminary cytogenetic data for a dose-response relationship of the irradiated blood lymphocytes. The data strongly indicate the existence of significant linear (α) and quadratic (β) components and are consistent with those published for the production of chromosome aberrations in comparable absorbed dose ranges.

Uncovering Proximity of Chromosome Territories using Classical Algebraic Statistics

Exchange type chromosome aberrations (ETCAs) are rearrangements of the genome that occur when chromosomes break and the resulting fragments rejoin with fragments from other chromosomes or from other regions within the same chromosome. ETCAs are commonly observed in cancer cells and in cells exposed to radiation. The frequency of these chromosome rearrangements is correlated with their spatial proximity, therefore it can be used to infer the three dimensional organization of the genome. Extracting statistical significance of spatial proximity from cancer and radiation data has remained somewhat elusive because of the sparsity of the data. We here propose a new approach to study the three dimensional organization of the genome using algebraic statistics. We test our method on a published data set of irradiated human blood lymphocyte cells. We provide a rigorous method for testing the overall organization of the genome, and in agreement with previous results we find a random relative positioning of chromosomes with the exception of the chromosome pairs {1,22} and {13,14} that have a significantly larger number of ETCAs than the rest of the chromosome pairs suggesting their spatial proximity. We conclude that algebraic methods can successfully be used to analyze genetic data and have potential applications to larger and more complex data sets.

Radiation Induced Lymphocyte Apoptosis: An Effective Way of “Tailoring” Radiotherapy to the Right Patients Only?

Radiation Induced Lymphocyte Apoptosis: An Effective Way of “Tailoring” Radiotherapy to the Right Patients Only?

A prospective study on histone γ-H2AX and 53BP1 foci expression in rectal carcinoma patients: correlation with radiation therapy-induced outcome.

BackgroundThe prognostic value of histone γ-H2AX and 53BP1 proteins to predict the radiotherapy (RT) outcome of patients with rectal carcinoma (RC) was evaluated in a prospective study. High expression of the constitutive histone γ-H2AX is indicative of defective DNA repair pathway and/or genomic instability, whereas 53BP1 (p53-binding protein 1) is a conserved checkpoint protein with properties of a DNA double-strand breaks sensor.MethodsUsing fluorescence microscopy, we assessed spontaneous and radiation-induced foci of γ-H2AX and 53BP1 in peripheral blood mononuclear cells derived from unselected RC patients (n = 53) undergoing neoadjuvant chemo- and RT. Cells from apparently healthy donors (n = 12) served as references.ResultsThe γ-H2AX assay of in vitro irradiated lymphocytes revealed significantly higher degree of DNA damage in the group of unselected RC patients with respect to the background, initial (0.5 Gy, 30 min) and residual (0.5 Gy and 2 Gy, 24 h post-radiation) damage compared to the control group. Likewise, the numbers of 53BP1 foci analyzed in the samples from 46 RC patients were significantly higher than in controls except for the background DNA damage. However, both markers were not able to predict tumor stage, gastrointestinal toxicity or tumor regression after curative RT. Interestingly, the mean baseline and induced DNA damage was found to be lower in the group of RC patients with tumor stage IV (n = 7) as compared with the stage III (n = 35). The difference, however, did not reach statistical significance, apparently, because of the limited number of patients.ConclusionsThe study shows higher expression of γ-H2AX and 53BP1 foci in rectal cancer patients compared with healthy individuals. Yet the data in vitro were not predictive in regard to the radiotherapy outcome.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1890-9) contains supplementary material, which is available to authorized users.

Correlation between radiation dose and p53 protein expression levels in human lymphocytes.

The aim of this research was to evaluate the relationship between p53 protein levels and absorbed doses from in vitro irradiated human lymphocytes. For this, samples of blood from 23 donors were irradiated with 0.5; 1; 2; and 4 Gy from a Cobalt-60 source, and the percentages of lymphocytes expressing p53 were scored using Flow Cytometry. The subjects were divided into 3 groups, in accordance with the p53 levels expressed per radiation dose: low (Group I), high (Group II), and excessive levels (Group III). For all groups, the analyses showed that the p53 expression levels increase with the absorbed dose. Particularly for groups I and II, the correlation between this protein expression and the dose follows the linear-quadratic model, such as for radioinduced chromosomal aberrations. In conclusion, our findings indicate possible applications of this approach in evaluating individual radiosensitivity prior to radiotherapeutical procedures as well as in medical surveillance of occupationally exposed workers. Furthermore, due to the rapidity of flow-cytometric analyses, the methodology here employed would play an important role in emergency responses to a large-scale radiation incident where many people may have been exposed.

Alpha particles induce pan-nuclear phosphorylation of H2AX in primary human lymphocytes mediated through ATM

The use of high linear energy transfer radiations in the form of carbon ions in heavy ion beam lines or alpha particles in new radionuclide treatments has increased substantially over the past decade and will continue to do so due to the favourable dose distributions they can offer versus conventional therapies. Previously it has been shown that exposure to heavy ions induces pan-nuclear phosphorylation of several DNA repair proteins such as H2AX and ATM in vitro. Here we describe similar effects of alpha particles on ex vivo irradiated primary human peripheral blood lymphocytes. Following alpha particle irradiation pan-nuclear phosphorylation of H2AX and ATM, but not DNA-PK and 53BP1, was observed throughout the nucleus. Inhibition of ATM, but not DNA-PK, resulted in the loss of pan-nuclear phosphorylation of H2AX in alpha particle irradiated lymphocytes. Pan-nuclear gamma-H2AX signal was rapidly lost over 24h at a much greater rate than foci loss. Surprisingly, pan-nuclear gamma-H2AX intensity was not dependent on the number of alpha particle induced double strand breaks, rather the number of alpha particles which had traversed the cell nucleus. This distinct fluence dependent damage signature of particle radiation is important in both the fields of radioprotection and clinical oncology in determining radionuclide biological dosimetry and may be indicative of patient response to new radionuclide cancer therapies.

Radioprotective Effect of Achillea millefolium L Against Genotoxicity Induced by Ionizing Radiation in Human Normal Lymphocytes.

The radioprotective effect of Achillea millefolium L (ACM) extract was investigated against genotoxicity induced by ionizing radiation (IR) in human lymphocytes. Peripheral blood samples were collected from human volunteers and incubated with the methanolic extract of ACM at different concentrations (10, 50, 100, and 200 μg/mL) for 2 hours. At each dose point, the whole blood was exposed in vitro to 2.5 Gy of X-ray and then the lymphocytes were cultured with mitogenic stimulation to determine the micronuclei in cytokinesis-blocked binucleated cell. Antioxidant capacity of the extract was determined using free radical-scavenging method. The treatment of lymphocytes with the extract showed a significant decrease in the incidence of micronuclei binucleated cells, as compared with similarly irradiated lymphocytes without any extract treatment. The maximum protection and decrease in frequency of micronuclei were observed at 200 μg/mL of ACM extract which completely protected genotoxicity induced by IR in human lymphocytes. Achillea millefolium extract exhibited concentration-dependent radical-scavenging activity on 1,1-diphenyl-2-picryl hydrazyl free radicals. These data suggest that the methanolic extract of ACM may play an important role in the protection of normal tissues against genetic damage induced by IR.

Antitumor Enhancement by Irradiated Haploidentical Donor Lymphocyte Infusion of Mice with Melanoma

Background: Previous researches have reported that donor lymphocyte infusion (DLI) provides a new approach for the treatment of hematological malignancies and some solid tumors. The present study was designed to discuss the antitumor effect on mice with melanoma and possible involvement of the mechanism of haploidentical DLI in CB6F1 mice→CC3HF1 mice (F1→F1) mouse model. Methods: An F1→F1 haploidentical infusion model was established. CB6F1 mice (H-2b/d) bearing melanoma were used as recipients. CC3HF1 mice (H-2d/k) were used as donors. Changes in tumor volume and mice survival, host-derived lymphocytes proliferation, cytotoxicity, donor cell survival in vivo, histopathological examination of important organs, and the secretion Th1/Th2 cytokines were analyzed. Results: Irradiated haploidentical DLI combined with low-dose cyclophosphamide (Cy) chemotherapy induced an antitumor effect on mice with melanoma using the F1→F1 infusion model. Graft-versus-host disease (GvHD) was not obvious in any DLI-treated groups. Donor lymphocytes disappeared within 5 days after infusion, while the antitumor effect continued to be observed. Moreover, the DLI-treated groups showed a significant increase in the secretion of Th1 cytokines, including IFN-γ and IL-2, and an enhanced proliferation of CD8⁺ T lymphocytes and NK cells. Conclusions: Irradiated haploidentical DLI without bone marrow transplantation offers a safe, feasible, and effective approach in the treatment of melanoma.