Bingrong Zhang

Response patterns of cytokines/chemokines in two murine strains after irradiation

PURPOSE To determine the plasma concentrations of acute responding cytokines/chemokines following 9-Gy ionizing radiation in C57BL/6 (radiation tolerant) and C3H/HeN (radiation sensitive) murine strains. METHODS AND MATERIALS Mice (5/group) received 9-Gy total body irradiation (TBI), and the plasma from each mouse was collected at 6h or 1, 2, 4, or 10 days after TBI. A multiplex bead array was used to assess the levels of 32 cytokines/chemokines in plasma to determine their common and strain-specific temporal responses. RESULTS The plasma levels of five cytokines/chemokines (Axl, FasL, ICAM-1, TARC, and TSLP) were beyond the detectable level. Five (VEGF, IL-2, IL-5, IL-17, and CD30) were unaffected by irradiation in either strain. Temporal patterns were similar in both murine strains for 10 of the cytokines tested, including G-CSF, IL-6, TCA-3, MCP-1, MIP-1γ, KC, CXCL 13, CXCL 16, MDC, and TIMP-1; the other 12 molecules (GM-CSF, IL-3, SCF, IL-1β, IL-4, IL-10, IL-12p70, MIP-1α, Eotaxin, TNF-α, sTNF-R1, and CD40) showed strain-specific response patterns. While a number of cytokines had temporal response patterns following TBI, the strains exhibited quantitatively different results. CONCLUSIONS The levels of 27 of the 32 plasma cytokines measured indicate the following: (1) different cytokine concentrations and temporal patterns in the two strains may partly explain different radiation sensitivities and sequelae following irradiation; (2) many of the cytokines/chemokines exhibit similar temporal responses in the two strains. These responses suggest the potential value of using a panel of cytokine/chemokine temporal patterns for radiation dosimetry. Although radiation doses will be difficult to quantitate due to the large variation in levels and temporal responses exhibited in the two murine strains, serial measurements of cytokines might help identify subjects exposed to radiation.

Triptolide Mitigates Radiation-Induced Pulmonary Fibrosis

Triptolide (TPL) may mitigate radiation-induced late pulmonary side effects through its inhibition of global pro-inflammatory cytokines. In this study, we evaluated the effect of TPL in C57BL/6 mice, the animals were exposed to radiation with vehicle (15 Gy), radiation with TPL (0.25 mg/kg i.v., twice weekly for 1, 2 and 3 months), radiation and celecoxib (CLX) (30 mg/kg) and sham irradiation. Cultured supernatant of irradiated RAW 264.7 and MLE-15 cells and lung lysate in different groups were enzyme-linked immunosorbent assays at 33 h. Respiratory rate, pulmonary compliance and pulmonary density were measured at 5 months in all groups. The groups exposed to radiation with vehicle and radiation with TPL exhibited significant differences in respiratory rate and pulmonary compliance (480 ± 75/min vs. 378 ± 76/min; 0.6 ± 0.1 ml/cm H2O/p kg vs. 0.9 ± 0.2 ml/cm H2O/p kg). Seventeen cytokines were significantly reduced in the lung lysate of the radiation exposure with TPL group at 5 months compared to that of the radiation with vehicle group, including profibrotic cytokines implicated in pulmonary fibrosis, such as IL-1β, TGF- β1 and IL-13. The radiation exposure with TPL mice exhibited a 41% reduction of pulmonary density and a 25% reduction of hydroxyproline in the lung, compared to that of radiation with vehicle mice. The trichrome-stained area of fibrotic foci and pathological scaling in sections of the mice treated with radiation and TPL mice were significantly less than those of the radiation with vehicle-treated group. In addition, the radiation with TPL-treated mice exhibited a trend of improved survival rate compared to that of the radiation with vehicle-treated mice at 5 months (83% vs. 53%). Three radiation-induced profibrotic cytokines in the radiation with vehicle-treated group were significantly reduced by TPL treatment, and this partly contributed to the trend of improved survival rate and pulmonary density and function and the decreased severity of pulmonary fibrosis at 5 months. Our findings indicate that TPL could be a potential new agent to mitigate radiation-induced pulmonary fibrosis.

In Vitro Sirius Red Collagen Assay Measures the Pattern Shift from Soluble to Deposited Collagen

In this study, we compared two in vitro collagen production assays ([(3)H]-proline incorporation and Sirius Red) for their ability to determine the pattern shift from soluble to deposited collagen. The effect of the antifibrotic agent, triptolide (TPL), on collagen production was also studied. The results showed that: (1) 48 h after NIH 3T3 (murine embryo fibroblast) and HFL-1(human fetal lung fibroblast) were exposed to transforming growth factor-beta 1 (TGF-β), there was an increase in soluble collagen in the culture medium; (2) on day 4, soluble collagen declined, whereas deposited collagen increased; (3) Sirius Red was easier to use than [(3)H]-proline incorporation and more consistently reflected the collagen pattern shift from soluble to deposited; (4) the in vitro Sirius Red assay took less time than the in vivo assay to determine the effect of TPL. Our results suggest that: (a) the newly synthesized soluble collagen can sensitively evaluate an agents capacity for collagen production and (b) Sirius Red is more useful than [(3)H]-proline because it is easier to use, more convenient, less time consuming, and does not require radioactive material.

Fibroblast Growth Factor-Peptide Promotes Bone Marrow Recovery After Irradiation

Various members of the fibroblast growth factor (FGF) family mitigate radiation-induced damage. We designed and synthesized the binding domain peptide of FGF-2 (FGF-P) with a dimer form resistant to peptidase and examined its mitigatory effect on murine bone marrow cells. NIH Swiss mice were exposed to different doses of total body irradiation (TBI) and treated with ten doses of 5 mg/kg FGF-P. We achieved the following results: (1) FGF-P stimulated the growth of bone marrow cells harvested from mice exposed to 3 Gy; (2) on day 25 after 6 Gy TBI, the number of leukocytes and granulocytes was higher in the FGF-P group than in the vehicle-alone group; (3) FGF-P significantly increased the number of pro-B and pre-B cells; and (4) FGF-P treatment in vivo increased the long-term hematopoietic stem cells (LT-HSC) in bone marrow. These data reveal the underlying mechanism by which FGF-P rescued a significant percentage of the exposed mice. The increase of LT-HSC in bone marrow leads to a concomitant increase of pro-B and pre-B cells followed by leukocytes and granulocytes, which in turn enhance immunity against infection.

Fibroblast growth factor-peptide improves barrier function and proliferation in human keratinocytes after radiation.

PURPOSE Epidermal keratinocytes, which can be severely damaged after ionizing radiation (IR), are rapid turnover cells that function as a barrier, protecting the host from pathogenic invasion and fluid loss. We tested fibroblast growth factor-peptide (FGF-P), a small peptide derived from the receptor-binding domain of FGF-2, as a potential mitigator of radiation effects via proliferation and the barrier function of keratinocytes. METHODS AND MATERIALS Keratinocytes isolated from neonatal foreskin were grown on transwells. After being exposed to 0, 5, or 10 Gy IR, the cells were treated with a vehicle or FGF-P. The permeability of IR cells was assessed by using transepithelial electrical resistance (TEER) and a paracellular tracer flux of fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA) with Ussing chambers. The cell proliferation was measured with yellow tetrazolium salt (MTT) and tritiated thymidine ([3H]-TdR) assays. The phosphorylation of extracellular signal-regulated kinases (ERK) was measured in an enzyme-linked immunosorbent (ELISA)-like assay, and the proteins related to tight junctions (TJ) and adherens junctions (AJ) were examined with Western blotting. We used a mouse model to assess the ability of FGF-P to promote the healing of skin β burns created with a strontium applicator. RESULTS We found (1) FGF-P reduced the permeability of irradiated keratinocytes, as evidenced by increased TEER and decreased diffusion of FITC-BSA, both associated with the regulation of different proteins and levels of TJ and AJ; and (2) FGF-P enhanced the proliferation of irradiated keratinocytes, as evidenced by increased MTT activity and [3H]-TdR incorporation, which was associated with activation of the ERK pathway; and (3) FGF-P promoted the healing of skin β burns. CONCLUSIONS FGF-P enhances the barrier function, including up-regulation of TJ proteins, increases proliferation of human keratinocytes, and accelerates the healing of skin β burns. FGF-P is a promising mitigator that improves the proliferation and barrier function of keratinocytes after IR.

Transition pattern and mechanism of B-lymphocyte precursors in regenerated mouse bone marrow after subtotal body irradiation.

Little is known about the effects of ionizing radiation on the transition and the related signal transduction of progenitor B cells in the bone marrow. Thus, using an NIH Swiss mouse model, we explored the impact of ionizing radiation on the early stage of B-cell development via an examination of the transition of CLP to pro-B to pre-B cells within bone marrow as a function of radiation doses and times. Our results showed that while the total number of bone marrow lymphoid cells at different stages were greatly reduced by subtotal body irradiation (sub-TBI), the surviving cells continued to transition from common lymphoid progenitors to pro-B and then to pre-B in a reproducible temporal pattern. The rearrangement of the immunoglobulin heavy chain increased significantly 1–2 weeks after irradiation, but no change occurred after 3–4 weeks. The rearrangement of the immunoglobulin light chain decreased significantly 1–2 weeks after sub-TBI but increased dramatically after 3–4 weeks. In addition, several key transcription factors and signaling pathways were involved in B-precursor transitions after sub-TBI. The data indicate that week 2 after irradiation is a critical time for the transition from pro-B cells to pre-B cells, reflecting that the functional processes for different B-cell stages are well preserved even after high-dose irradiation.

A New Flavonoid Regulates Angiogenesis and Reactive Oxygen Species Production

The tumor vascular system, which is critical to the survival and growth of solid tumors, has been an attractive target for anticancer research. Building on studies that show that some flavonoids have anticancer vascular effects, we developed and analyzed the flavonoid derivative R24 [3, 6-bis (2-oxiranylmethoxy)-9H-xanthen-9-one]. A CAM assay revealed that R24 disrupted neovascular formation; fewer dendrites were detected and overall dendritic length was shorter in the R24-treated chicken embryos. The antiproliferative effect of R24 was measured by MTT assay in A549 (lung cancer), AsPC-1 (pancreatic cancer), HCT-116 (colorectal cancer), and PC-3 (prostate cancer) cell lines. R24 reduced proliferation with an IC50 of 3.44, 3.59, 1.22, and 11.83 μM, respectively. Cell-cycle analysis and Annexin-V/propidium iodide staining showed that R24 induced apoptosis. In addition, R24 regulated intracellular ROS production in a dose-dependent manner. CM-H2DCFDA staining indicated that intracellular ROS production increased with the R24 dose. In summary, we found that R24 exhibits potent antiangiogenic and antiproliferative effects, induces apoptosis, and promotes ROS production.

Radiation-Induced Elevation of Plasma DNA in Mice is Associated with Genomic Background

Genomic background helps determine sensitivity to TBI. Since the LD50/30 (the dose that causes death in half of exposed mice within 30 days) following TBI varies between murine strains, we tested four murine strains to determine whether TBI sensitivity was associated with different levels of circulating DNA after radiation. The LD50/30 for the mice we tested – BALB/c, NIH Swiss, C3H/HeN, and C57BL/6 – is approximately 5.8 ± 0.3, 7.3 ± 0.2, 7.4 ± 0.3, and 8.5 ± 0.3 Gy, respectively. We estimated the radiation dose at which circulating DNA reached a peak level (peakGy), and mice were subjected to different doses at 1.84 Gy/min. The peakGy was 6, 7, 9, and 9 Gy for BALB/c, NIH Swiss, C3H/HeN, and C57BL/6, which corresponds to each strain’s respective LD50/30. BALB/c, the most sensitive strain, had the lowest DNA concentration at peakGy, while C57BL/6, the most resistant strain, had the highest concentration at peakGy. At 7 Gy, the plasma DNA was approximately 3,754 ± 636, 8,238 ± 2,704, 9,773 ± 2,821, and 22,733 ± 5,914 ng/ml for BALB/c, NIH Swiss, C3H/HeN, and C57BL/6, respectively. These findings support our hypothesis that plasma DNA level is associated with genomic background.

The role of mitochondrial proteomic analysis in radiological accidents and terrorism.

In the wake of the 9/11 terrorist attacks and the recent Level 7 nuclear event at the Fukushima Daiichi plant, there has been heightened awareness of the possibility of radiological terrorism and accidents and the need for techniques to estimate radiation levels after such events. A number of approaches to monitoring radiation using biological markers have been published, including physical techniques, cytogenetic approaches, and direct, DNA-analysis approaches. Each approach has the potential to provide information that may be applied to the triage of an exposed population, but problems with development and application of devices or lengthy analyses limit their potential for widespread application. We present a post-irradiation observation with the potential for development into a rapid point-of-care device. Using simple mitochondrial proteomic analysis, we investigated irradiated and nonirradiated murine mitochondria and identified a protein mobility shift occurring at 2-3 Gy. We discuss the implications of this finding both in terms of possible mechanisms and potential applications in bio-radiation monitoring.

Alteration of plasma galactose/N-acetylgalactosamine level after irradiation.

Although glycoproteins possess a variety of functional and structural roles in intracellular and intercellular activities, the effect of ionizing radiation (IR) on glycosylation is largely unknown. To explore this effect, we established a sandwich assay in which PHA-L, a phytohaemagglutinin that agglutinates leukocytes, was used as a coating layer to capture glycoproteins containing complex oligosaccharides; the bound glycoproteins were then measured. C57BL/6 mice were exposed to 0, 3, 6, or 10 Gy, and the plasma was collected at 6, 12, 18, 24, 48, 72, or 168 h and then analyzed for galactose/N-acetylgalactosamine (Gal/GalNAc) containing proteins. We found that (1) the sandwich assay accurately measured the level of glycoproteins, (2) 6-12 h after IR, the amount of glycoproteins containing GalNAc increased, and (3) at 72 and 168 h, 10 Gy was associated with a decrease in Gal/GalNAc. These IR-induced alterations might relate to the release of glycoproteins into the blood and the damage of the proteins and genes that are related to the glycosylation process.