Chi-Shiun Chiang

Induction of acute phase gene expression by brain irradiation.

PURPOSE To investigate the in vivo acute phase molecular response of the brain to ionizing radiation. METHODS AND MATERIALS C3Hf/Sed/Kam mice were given midbrain or whole-body irradiation. Cerebral expression of interleukins (IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6), interferon (IFN-gamma), tumor necrosis factors (TNF-alpha and TNF-beta), intercellular adhesion molecule-1 (ICAM-1), inducible nitric oxide synthetase (iNOS), von Willebrand factor (vWF), alpha 1-antichymotrypsin (EB22/5.3), and glial fibrillary acidic protein (GFAP) was measured at various times after various radiation doses by ribonuclease (RNase) protection assay. The effects of dexamethasone or pentoxifylline treatment of mice on radiation-induced gene expression were also examined. RESULTS Levels of TNF-alpha, IL-1 beta, ICAM-1, EB22/5.3 and to a lesser extent IL-1 alpha and GFAP, messenger RNA were increased in the brain after irradiation, whether the dose was delivered to the whole body or only to the midbrain. Responses were radiation dose dependent, but were not found below 7 Gy; the exception being ICAM-1, which was increased by doses as low as 2 Gy. Most responses were rapid, peaking within 4-8 h, but antichymotrypsin and GFAP responses were delayed and still elevated at 24 h, by which time the others had subsided. Pretreatment of mice with dexamethasone or pentoxifylline suppressed radiation-induced gene expression, either partially or completely. Dexamethasone was more inhibitory than pentoxifylline at the doses chosen. CONCLUSIONS The initial response of the brain to irradiation involves expression of inflammatory gene products, which are probably responsible for clinically observed early symptoms of brain radiotherapy. This mechanism explains the beneficial effects of the clinical use of steroids in such circumstances.

A sense of danger from radiation.

Abstract McBride, W. H., Chiang, C-S., Olson, J. L., Wang, C-C., Hong, J-H., Pajonk, F., Dougherty, G. J., Iwamoto, K. S., Pervan, M. and Liao, Y-P. A Sense of Danger from Radiation. Radiat. Res. 162, 1–19 (2004). Tissue damage caused by exposure to pathogens, chemicals and physical agents such as ionizing radiation triggers production of generic “danger” signals that mobilize the innate and acquired immune system to deal with the intrusion and effect tissue repair with the goal of maintaining the integrity of the tissue and the body. Ionizing radiation appears to do the same, but less is known about the role of “danger” signals in tissue responses to this agent. This review deals with the nature of putative “danger” signals that may be generated by exposure to ionizing radiation and their significance. There are a number of potential consequences of “danger” signaling in response to radiation exposure. “Danger” signals could mediate the pathogenesis of, or recovery from, radiation damage. They could alter intrinsic cellular radiosensitivity or initiate radioadaptive responses to subsequent exposure. They may spread outside the locally damaged site and mediate bystander or “out-of-field” radiation effects. Finally, an important aspect of classical “danger” signals is that they link initial nonspecific immune responses in a pathological site to the development of specific adaptive immunity. Interestingly, in the case of radiation, there is little evidence that “danger” signals efficiently translate radiation-induced tumor cell death into the generation of tumor-specific immunity or normal tissue damage into autoimmunity. The suggestion is that radiation-induced “danger” signals may be inadequate in this respect or that radiation interferes with the generation of specific immunity. There are many issues that need to be resolved regarding “danger” signaling after exposure to ionizing radiation. Evidence of their importance is, in some areas, scant, but the issues are worthy of consideration, if for no other reason than that manipulation of these pathways has the potential to improve the therapeutic benefit of radiation therapy. This article focuses on how normal tissues and tumors sense and respond to danger from ionizing radiation, on the nature of the signals that are sent, and on the impact on the eventual consequences of exposure.

Reactive Gliosis as a Consequence of Interleukin-6 Expression in the Brain: Studies in Transgenic Mice

Gliosis is a characteristic pathologic state in many CNS disorders. Cytokines are considered to be effectors of gliosis. In order to explore the role of IL-6 in gliosis, the temporal and spatial expression of the IL-6 gene and its consequent effects on the brain were studied in a GFAP-IL6 transgenic mouse model. In GFAP-IL6 mice, IL-6 transgene expression was detectable in the brain at 1 week postnatally and increased to maximal levels by 3 months of age before declining at 8 and 12 months. Enhanced glial fibrillary acidic protein (GFAP) (marker for astrocytes) and Mac-I (marker for microglia) mRNA expression were first prominent at 1 month, increased to maximum levels by 3 months and remained significantly elevated through 12 months of age. Western blot analysis revealed that the enhanced GFAP mRNA expression in these transgenic mice was accompanied by increased GFAP protein levels. Immunostaining for Mac-I demonstrated that in addition to an increased staining intensity, the number of cells expressing the microglial/macrophage marker was also apparently increased, particularly in the cerebellum and brain stem. Concurrent with IL-6 transgene mRNA expression and reactive gliosis, upregulation of IL-1 alpha/beta, TNF alpha, ICAM-1 and EB22/5.3 (acute-phase reactant) but not inducible nitric oxide synthase gene expression was also observed. EB22/5.3 mRNA expression was most prominent and increased progressively with age. Expression of the IL-6, GFAP and EB22/5.3 RNAs was found to have similar distribution in the brain being found predominantly in the cerebellum, brain stem and sub-cortical regions. In conclusion, the constitutive expression of IL-6 in the brain induced the development of a pronounced and lifelong reactive gliosis affecting both astrocytes and microglia. The altered state of these cells may contribute to the functional and structural CNS impairment exhibited by the GFAP-IL6 mice. Finally, in these mice, expression of the EB22/5.3 gene correlated closely with the progression of neuropathy indicating that this acute-phase response gene was a good marker for and may be involved in the pathogenesis of CNS injury mediated by the expression of IL-6.

Delayed molecular responses to brain irradiation

The chance of life-threatening complications occurring late after brain irradiation limits the efficacy of this form of cancer therapy. The molecular and cellular events that trigger radiation-induced brain damage are still unknown, but since they have the potential to serve as valuable targets for therapeutic intervention they are worth delineating. In this murine study, the effect of irradiation on the expression of molecules which are known to contribute to brain damage in other model systems was examined. Expression of genes encoding cytokines (TNF-alpha/beta, IL-1 alpha/beta, IL-2, IL-3, IL-4, IL-5, IL-6 and IFN-gamma), cytokine receptors (TNF-Rp55 and p75, IL-1R- p60 and p80, IFN-gamma R, and IL-6R), the cell adhesion molecule (ICAM-1), inducible nitric oxide synthetase (iNOS), anti-chymotrypsin (EB22/5.3), and the gliotic marker (GFAP) was evaluated over a 6-month period using a sensitive RNase protection assay (RPA). We had previously demonstrated that within 24 h of brain irradiation there is an acute transitory molecular response involving TNF-alpha, IL-1, ICAM-1, EB22/5.3 and GFAP. This study shows re-elevation of TNF-alpha, EB22/5.3 and GFAP mRNA levels at 2-3 months, but only TNF-alpha mRNA was overexpressed at 6 months. These time points are when neurological abnormalities are seen after higher doses. The data suggest that TNF-alpha may be involved in late brain responses to irradiation and could contribute to clinical symptoms.

Radiation-induced astrocytic and microglial responses in mouse brain.

The aim of this study was to investigate the responses of astrocytes and microglia to whole brain irradiation. Levels of glial fibrillary acidic protein (GFAP), which is a marker for astrocytes, were measured by ELISA in irradiated brains taken at varying time points after irradiation. GFAP levels were increased between 120 and 180 days after single doses of 20-45 Gy radiation, but not after lower doses (2 or 8 Gy). The increases in GFAP levels were confirmed by Western blot analysis and immunohistochemical staining which showed that the number of GFAP-positive astrocytes was increased, as was their staining intensity. Coincidently with the increase in astrocyte staining, there was an increase in the number and the intensity of microglial cell staining for Mac I antigen. Autoradiography of brain tissue following in vivo administration of [3H]thymidine showed an increased number of labelled cells during the same time period. The radiation-induced astrocytic and microglial responses that follows brain irradiation is indicative of reactive gliosis and inflammation occurring during the latent period up to the onset of late radiation-induced injury. This gliosis increases with radiation dose. The possibility that gliosis may participate in modifying postirradiation injury in the brain is discussed.

Rapid induction of cytokine gene expression in the lung after single and fractionated doses of radiation.

PURPOSE To investigate cytokine gene expression in the lung after single and fractionated doses of radiation, and to investigate the effect of steroids and the genetic background. MATERIALS AND METHODS Expression of cytokine genes (mTNF-alpha, mIL-1alpha, mIL-1beta, mIL-2, mIL-3, mIL-4, mIL-5, mIL-6, mIFN-gamma) in the lungs of C3H/HeJ and C57BL/6J mice was measured by RNase protection assay at different times after various doses of radiation. The effects of dexamethasone and fractionated radiation treatment on gene expression were also studied. RESULTS IL-1beta was the major cytokine induced in the lungs of C3H/HeJ mice within the first day after thoracic irradiation. Radiation doses as low as 1 Gy were effective. Responses to 20 Gy irradiation peaked within 4-8h and subsided by 24 h. With the exception of IL-1alpha and TNF-alpha, the other cytokines that were investigated had undetectable pre-treatment mRNA levels and were not radiation inducible. Similar responses were seen in C57BL/6J mice, although TNF-alpha was induced and there were some quantitative differences. Pre-treatment of C3H/HeJ mice with dexamethasone reduced basal and induced IL-1 levels, but complete inhibition was not achieved. Dexamethasone was also effective if given immediately after irradiation. Fractionated daily doses of radiation (4 Gy/day) helped to maintain cytokine gene expression for a longer period. CONCLUSIONS Inflammatory genes are rapidly induced in the lung by irradiation. This response cannot be readily abolished by steroid pre-treatment. Fractionated treatment schedules help to perpetuate the response.

Designing Multi-Branched Gold Nanoechinus for NIR Light Activated Dual Modal Photodynamic and Photothermal Therapy in the Second Biological Window

Gold nanoechinus can sensitize the formation of singlet oxygen in the first and the second near-infra red (NIR) biological windows and exert in vivo dual modal photodynamic and photothermal therapeutic effects (PDT and PTT) to destruct the tumors completely. This is the first literature example of the destruction of tumors in NIR window II induced by dual modal nanomaterial-mediated photodynamic and photothermal therapy (NmPDT & NmPTT).

Preparation of Fluorescent Magnetic Nanodiamonds and Cellular Imaging

Magnetic nanodiamonds were prepared via solid-state microwave arcing of a nanodiamond-ferrocene mixed powder in a focused microwave oven. High-resolution transmission electron microscope (HRTEM) images show that a magnetic nanodiamond is composed of iron nanoparticles encapsulated by graphene layers on the surface of nanodiamonds. Fluorescence property was introduced onto magnetic nanodiamonds by chemical modification of magnetic nanodiamonds via surface grafting of poly(acrylic acids) and fluorescein o-methacrylate. Fluorescent magnetic nanodiamonds are water soluble with a solubility of approximately 2.1 g/L. Cellular-imaging experiments show that fluorescent magnetic nanodiamonds could be ingested by HeLa cells readily in the absence of agonist (i.e., folate) moieties on the surface of nanodiamonds.

Macrophage/microglial-mediated primary demyelination and motor disease induced by the central nervous system production of interleukin-3 in transgenic mice.

Activated macrophage/microglia may mediate tissue injury in a variety of CNS disorders. To examine this, transgenic mice were developed in which the expression of a macrophage/microglia activation cytokine, interleukin-3 (IL-3), was targeted to astrocytes using a murine glial fibrillary acidic protein fusion gene. Transgenic mice with low levels of IL-3 expression developed from 5 mo of age, a progressive motor disorder characterized at onset by impaired rota-rod performance. In symptomatic transgenic mice, multi-focal, plaque-like white matter lesions were present in cerebellum and brain stem. Lesions showed extensive primary demyelination and remyelination in association with the accumulation of large numbers of proliferating and activated foamy macrophage/microglial cells. Many of these cells also contained intracisternal crystalline pole-like inclusions similar to those seen in human patients with multiple sclerosis. Mast cells were also identified while lymphocytes were rarely, if at all present. Thus, chronic CNS production of low levels of IL-3 promotes the recruitment, proliferation and activation of macrophage/microglial cells in white matter regions with consequent primary demyelination and motor disease. This transgenic model exhibits many of the features of human inflammatory demyelinating diseases including multiple sclerosis and HIV leukoencephalopathy.

Radiation enhances tumor necrosis factor α production by murine brain cells

Abstract Astrocytes and microglial cells cultured from murine brain were stimulated to produce tumor necrosis α (TNF) by exposure to lipopolysaccharide (LPS). TNF α production began within 2 h with maximum production between 4 and 8 h after atimulation. Clinically relevant low (2 Gy), but not high (8 Gy), doses of radiation significantly increased TNF production by astrocytes and microglial cells in response to LPS. The radiation effect was even more marked with multiple 2 Gy doses. TNF is cytotoxic for oligodendrocytes and for certain tumor cells. It increases vascular permeability and enhances immune responses as well as having biological effects. It is conceivable that production of TNF by astrocytes and microglial cells during clinical radiation theraphy might influence the responses of tumor and/or normal CNS tissues.