pKC modulates integrin expression that contributes to fibrotic changes in irradiated thyroid tissue.

AIM: We hypothesized that radiation-induced fibrosis was, in part, a result of altered signal transduction that directly modulates integrin expression and may indirectly affect the extracellular matrix (ECM). Major focus was given on protein kinase C (pKC). MATERIALS AND METHODS: Rat FRTL-5 and primary thyroid cells were exposed to proton radiation (5 and 10 Gy). Hours to days after exposure, a series of assays were performed. In addition, the neck region of Lewis rats was proton-irradiated to 40 Gy (5 Gy/day or 10 Gy/day). At 11 weeks after exposure, thyroid tissue was evaluated. RESULTS: Accumulation of ECM in irradiated FRTL-5 and primary thyroid cells was coincident with loss of tissue organization and follicularization at one or more doses and time points. Several pKC isoforms increased post-irradiation, which coincided with modulated integrin expression; fibronectin, laminin and collagen were also altered (p<0.05 vs. 0 Gy). Modulation of thyroid cells in culture with 12-O-tetradecanoylphorbol-13-acetate (TPA)±calphostin C supported a direct role of pKC in these altered properties. Thyroid tissue from irradiated rats had significantly more fibrotic lesions and increases in several pKC isoforms, integrins and fibronectin compared to 0-Gy (p<0.05). CONCLUSION: pKC is a likely contributor to alteration of key players associated with radiation-induced fibrosis.

Effect of radiation and repeated sub-culturing on the transforming growth factor-ß1 signaling pathway in FRTL-5 cells.

BACKGROUND/AIM: Fisher rat thyroid cells (FRTL-5) display increased proliferation, reduced follicularization and decreased thyroxin release with repeated sub-culturing. These changes occur earlier and more rapidly following exposure to ionizing radiation. We hypothesized that altered transforming growth factor-ß1 (TGF-ß1) signaling contributes to these differences. MATERIALS AND METHODS: Assessments included FRTL-5 cell growth rate and quantification of TGF-ß1 ligand and receptors. The levels and activity of Smads2, 3 and 4 were measured by western blotting and the ability of TGF-ß1 to regulate cyclin A and plasminogen activator inhibitor type 1 (PAI-1) activity was assessed using transfection assays. RESULTS: TGF-ß1 production increased after radiation but returned to control levels after repeated sub-culturing. There was no difference in TGF-ß1 levels between un-irradiated cells at low versus high-passage number. TGF-ß1 receptors and basal levels of Smads2, 3 and 4 remained unchanged. However, there were significant changes in cell proliferation, TGF-ß1-mediated Smads2 and 3 activation and in TGF-ß1's ability to regulate cyclin A and PAI-1 transcription in irradiated and repeatedly sub-cultured cells (p<0.05). CONCLUSION: Collectively, these results support the conclusion that alterations in the TGF-ß1 pathway contribute to phenotypic changes in FRTL-5 cells as a function of passage number and radiation.

Strain-related differences and radiation quality effects on mouse leukocytes: gamma-rays and protons (with and without aluminum shielding).

Increasing evidence indicates that radiation-induced genomic instability plays an important role in the development of cancer. However, radiation quality and genetic background can influence the outcome. The goal of this study was to quantify radiation-induced changes in lymphocyte populations in mouse strains known to differ in susceptibility to genomic instability (C57BL/6, resistant; CBA/Ca, susceptible). The effects of whole-body exposure to γ-rays and protons, with and without aluminum shielding, were compared. Total radiation doses of 0, 0.1, 0.5, and 2.0 Gy were delivered and subsets of mice from each group were euthanized on days 1 and 30 after exposure for spleen and bone marrow analyses. In the spleen on day 1, lymphocyte counts were decreased (p<0.05) in C57, but not CBA, mice irradiated with 2 Gy. By day 30 in the C57 strain, counts were still low in the group exposed to 2 Gy shielded protons. Some strain- and radiation-dependent differences were also noted in percentages of specific lymphocyte populations (T, B, NK) and the CD4:CD8 ratio. In bone marrow, percentages of stem/progenitor cells (CD34(+), Ly-6A/E(+), CD34(+)Ly-6A/E(+)) were generally highest 1 day after 2 Gy irradiation, regardless of strain and radiation type. Based on dUTP incorporation, bone marrow cells from C57 mice had consistently higher levels of DNA damage on day 30 after irradiation with doses less than 2 Gy, regardless of quality. Annexin V binding supported the conclusion that C57 bone marrow cells were more susceptible to radiation-induced apoptosis. Overall, the data indicate that leukocytes of CBA mice are less sensitive to the effects of high-linear energy transfer radiation (shielded protons) than C57 mice, a phenomenon consistent with increased possibility for genomic instability and progression to a malignant cell phenotype after sublethal damage.

Effects of protons and HZE particles on glutamate transport in astrocytes, neurons and mixed cultures.

Radiation-induced neurotoxicity is a well-characterized phenomenon. However, the underlying mechanism of this toxicity is poorly understood. In the central nervous system (CNS), excitotoxic mechanisms are implicated in many neurodegenerative disease processes. Pivotal to the excitotoxic pathway is dysfunction of glutamate signaling. We reported previously that exposure to low-LET γ radiation results in altered glutamate transport in neurons and astrocytes. In the present study, we sought to investigate the effects of various particle radiations of differing LET on glutamate transport as a measure of the neurochemical vulnerability of the CNS. NTera2-derived neurons and astrocytes isolated as pure and mixed cultures were exposed to doses of 10 cGy, 50 cGy or 2 Gy of 250 MeV protons, 290 MeV/nucleon carbon ions, or 1000 MeV/nucleon iron ions. Transporter function was assessed at 3 h, 2 days and 7days after exposure. Functional assessment of glutamate transport revealed that neurons and astrocytes respond in a reciprocal manner after exposure to particle radiation. Uptake activity in neurons increased after particle irradiation. This effect was evident as late as our last time (7 days) after exposure (P < 0.05). In astrocytes, transporter activity decreased after exposure. The decrease in uptake observed in astrocytes was evident 7 days after exposure to carbon and iron ions. Uptake in mixed cultures after exposure to all three forms of radiation revealed a muted interactive response suggestive of the individual responses of each cellular phenotype acting in opposition.

Gene expression analysis of oxidative stress and apoptosis in proton-irradiated rat retina.

UNLABELLED: The aim of this study was to examine the induction of oxidative stress and apoptosis-associated gene expression profiles in retina after proton irradiation exposure at 0.5 to 4 Gy. MATERIALS AND METHODS: One eye of each Sprague-Dawley rat (6 per group) was irradiated with a conformal proton beam to total doses of 0, 0.5, 1 and 4 Gy. Retinal tissues were isolated for characterization of gene expression profiles 6 hours after proton radiation. RESULTS: For oxidative stress, many genes responsible for regulating the production of reactive oxygen species (ROS) were significantly up-regulated (Fmo2, Gpx2, Noxa1 and Sod3) compared to controls. Several important genes involved in the initiation or activation of apoptotic signaling pathways were significantly up-regulated following irradiation (Fas, Faslg, Trp63 and Trp73). TUNEL assay and caspase-3 immunocytochemical analysis revealed increased apoptotic immunoreactivity following irradiation. CONCLUSION: The data revealed that exposure to proton radiation induced oxidative stress-associated apoptosis. In response to ionizing radiation, the expression of genes involved in pathways mediating apoptosis may be differentially regulated in different dose regimens.

Alterations in glutamate uptake in NT2-derived neurons and astrocytes after exposure to gamma radiation.

Currently, the cellular and molecular mechanisms that underlie radiation-induced damage in the CNS are unclear. The present study began investigations of the underlying mechanism(s) for radiation-induced neurotoxicity by characterizing glutamate transport expression and function in neurons and astrocytes after exposure to gamma rays. NTera2-derived neurons and astrocytes, isolated as pure cultures, were exposed to doses of 10 cGy, 50 cGy and 2 Gy gamma rays, and transporter expression and function were assessed 3 h, 2 days and 7 days after exposure. In neurons, at 7 days after exposure, a significant increase was detected in EAAT3 after 50 cGy (P < 0.05) and a dose-dependent increase in GLT-1 expression was seen between doses of 10 and 50 cGy (P < 0.05). Functional assays of glutamate uptake revealed that neurons and astrocytes respond in a reciprocal manner after irradiation. Neurons responded to radiation exposure by increased glutamate uptake, an effect still evident at our last time (7 days) after exposure (P < 0.05). The astrocyte response to gamma radiation was an initial decrease in uptake followed by recovery to baseline levels at 2 days after exposure (P < 0.05). The observations made in this study demonstrate that neurons and astrocytes, while part of the same multifunctional unit, have distinct functional and reciprocal responses. The response in neurons appears to indicate a protracted response with potential long-term effects after irradiation.

In vitro effects of 3 common arthroscopic instruments on articular cartilage.

PURPOSE: The purpose of this study was to compare chondroplasty performed with an ExoJet high-pressure fluid-driven burr (Mitek, Norwood, MA), a mechanical shaver, and a bipolar radiofrequency (RF) wand on articular cartilage-covered condyles taken from sheep cadavers that were induced to have an osteoarthritic-like condition, and corresponding healthy control tissue. TYPE OF STUDY: Experimental designed animal cadaveric, biochemical, and histologic study. METHODS: Sheep condyles were used as a source of articular cartilage. Femurs were extracted approximately 1 hour postmortem and a transverse section of the condyles was made. Half of the samples were treated to induce an osteoarthritic-like condition. The condyles were then subjected to chondroplasty performed with the ExoJet high-pressure fluid-driven burr, a mechanical shaver, and a bipolar RF wand under sterile saline solution by an experienced orthopaedic surgeon. Twenty cross-sections from each condyle were examined by confocal microscopy to measure smoothness and depth of tissue damage to the articular cartilage caused by each of the 3 instruments. RESULTS: The ExoJet high-pressure fluid-driven burr and the bipolar RF wand left a smoother surface on the articular cartilage compared with the mechanical shaver. Additionally, the ExoJet fluid-burr caused slightly less tissue damage to the cartilage than the bipolar RF wand, both of which were less damaging than the shaver. CONCLUSIONS: Orthopaedists have multiple choices for surgical instruments used on cartilage. However, the effect on the integrity of the cartilage left remaining at the knee was previously unknown. Based on this study, a fluid-burr appears to leave the cartilage with a smaller zone of injury than does the RF wand or shaver. It also leaves the cartilage surface smoother than the shaver. During surgical procedures, minimizing cartilage breakdown and smooth remaining surfaces are desired because they minimize the vulnerable tissue to further destruction. A fluid burr leaves cartilage with less injury and with a smoother surface than do more traditional surgical instruments. CLINICAL RELEVANCE: This information should help surgeons in their selection of currently available surgical instruments and should aid engineers in the design of future instruments that function to modify articular cartilage.

Serum antiguanosine antibodies as a marker for SLE disease activity and pathogen potential.

BACKGROUND: This article reviews research conducted on the immunogenicity of the nucleosides of DNA, especially guanosine, the most immunologically active nucleoside. Discussed is the relationship between circulating antibodies to guanosine, their potential role in SLE disease activity, the binding properties of monoclonal antiguanosine antibody (4H2) compared to polyclonal antiguanosine antibodies in humans with SLE, cell membrane penetration by these antibodies and their interference with signal transduction possibly related to their binding to mitochondria and their apparent GTPase activity. METHODS: Enzyme-linked immunosorbent assay methodology was used to show clinical relationships between antiguanosine antibody levels and disease activity in SLE. These results are discussed along with methods of detecting cell penetration by this antibody using special staining techniques, laser-scanning microscope detection of mitochondrial localization, and interference of cAMP and pKA production/activation. Additionally, there is some discussion regarding the assay used to detect enzymatic activity of antiguanosine antibodies. RESULTS: Enhanced circulating levels of antiguanosine antibodies in patients with SLE correlate closely with SLE disease activity. Other factors are discussed that support the pathogenic potential of these antibodies, including their ability to penetrate lymphocytes, bind to mitochondria, inactivate mitochondrial function, interfere with signal transduction, and their potential enzymatically activity. CONCLUSIONS: Antiguanosine antibodies correlate with SLE disease activity and may be pathogenically important in SLE by interfering with signal transduction, inactivating mitochondrial and cell function in patients with SLE.

Shaver, bipolar radiofrequency, and saline jet instruments for cutting meniscal tissue: a comparative experimental study on sheep menisci.

PURPOSE: The purpose of this study was to compare the quality of meniscal tissue cut with 3 different surgical instruments (traditional shavers, bipolar radiofrequency (RF) wands, and a high-pressure saline jet) and that of control menisci. TYPE OF STUDY: Experimental design, biochemical and histologic study. METHODS: Sixty samples of sheep menisci were separated into 4 groups. Three groups were shaved on the apical surface with the different instruments. The smoothness of the cut surfaces was evaluated visually by an orthopaedic surgeon and then scored by laser scanning cytometry and by line measurement analysis. The depth of tissue damage was measured by fluorescent cytochemistry. Means and standard deviations were calculated and comparative statistics used (P < .05). RESULTS: The edges cut by the saline jet and bipolar RF were significantly smoother when judged by the surgeon than those cut by traditional shaver. There was no significant difference between the saline jet and bipolar RF. There were no significant differences in smoothness when measured by laser scanning cytometry or by line measurement techniques. The control menisci had less depth of damage along the edge as measured by fluorescent cytochemistry than did any of the menisci cut with the instrument. The saline jet had significantly less depth of damage than did the shaver. No other significant differences existed between the instruments for depth of damage. CONCLUSIONS: The results of our investigation conclude that high-pressure saline instruments may cause less damage to residual meniscal tissue when compared with bipolar RF and shavers. Saline jets and bipolar RF also produce a smoother cut than shavers. CLINICAL RELEVANCE: Surgeons may want to consider the degree of residual damage to meniscal tissue from the application of various surgical instruments. Saline jets may be a superior cutting instrument than RF or shavers when considering depth of residual damage and smoothness of residual meniscal edges.

Changes in the activation and reconstitution of lymphocytes resulting from total-body irradiation correlate with slowed tumor growth.

Alterations in cytokine secretion, activation marker expression, and immune cell concentrations were investigated at sequential time points following delivery of total-body irradiation (TBI) to C57BL/6 mice (n = 64) in the Lewis lung tumor model. Significantly slower tumor growth was observed when a 3-Gy dose of TBI was administered 2 h prior to tumor implantation (p < 0.05). The antitumor effect was correlated with an increased CD4:CD8 T cell ratio and heightened leukocyte blastogenesis. TBI was also found to induce an expansion of natural killer (NK) cells in the blood and spleen of tumor-bearing animals 10 days after irradiation (2.8 x 10(6) NK cells/spleen in test mice compared to 8.9 x 10(5) NK cells/spleen in normal control animals). However, no significant differences were found in NK cell levels within the tumor tissue. Enhanced production of interleukin (IL)-12 and IL-18 from spleen supernatants was consistent with an augmentation of the NK cell response. Significant reductions in transforming growth factor-beta1 (TGF-beta1) and vascular endothelial growth factor, both of which are associated with immune suppression, were also noted. Furthermore, TBI induced changes in expression of CD25 and CD71 activation markers, suggesting that radiation may alter tumor surveillance. Taken together, the relative percentages and activation status of immune cell compartments support the conclusion that these TBI-induced changes function to slow tumor progression.

IL-2 gene and antisense TGF-beta1 strategies counteract HSV-2 transformed tumor progression.

The H238 tumor cells are Herpes simplex virus type 2-transformed BALB/c mouse fibroblasts that constitutively express transforming growth factor (TGF-beta1). TGF-beta can diminish immune capacity, whereas interleukin 2 (IL-2) is stimulatory to the immune system and can counteract the negative effects of TGF-beta1. The H238-BALB/c system provides a syngeneic model to evaluate new strategies with the potential to ameliorate tumor-induced immune depression. Plasmids expressing either antisense TGF-beta1 or murine Il-2 were constructed and stably transformed cells generated (masH238 and H238-IL2, respectively). In vitro measurements (ELISA and RT-PCR) demonstrated a >70% decrease in TGF-beta1 secretion by the masH238 tumor cells, and significant levels of IL-2 production by the H238-IL2 transfected cells when compared to wild type and control plasmid-transfected H238 cells. BALB/c mice injected subcutaneously with the masH238 cells developed significantly smaller tumors than the controls. Mice injected with H238-IL-2 cells developed tumors that failed to progress relative to control tumor growth. The differences in tumor growth in the mice were associated with enhanced immune reactivity and an increased response to T lymphocyte mitogens. Significant differences were also noted in lymphocyte populations and expression of CD25 and CD71 activation markers in the blood and spleens of mice receiving transfected tumor cells. Collectively, the data demonstrate that strategies employing antisense TGF-beta1 and IL-2 expression by transfected tumor cells can counteract the progression of a TGF-beta1-secreting tumor and enhance immune function involving modulation of T lymphocyte populations.

Antiguanosine antibodies in murine and human lupus have the internal image of G-binding proteins.

OBJECTIVE: To examine the binding specificities of serum IgG antibodies of mouse and human origin directed against guanosine. The immunodominance of guanosine compared with the other nucleosides was established in the MRL/lpr murine model of systemic lupus erythematosus (SLE). Serum antiguanosine autoantibodies in human lupus correlate with nephritis and polyserositis in acute disease as well as in exacerbations of disease symptoms. METHODS: Antiguanosine autoantibodies obtained from humans with SLE were compared to a murine monoclonal antiguanosine antibody, 4H2. The fine specificity of the antiguanosine-binding site was determined by methylation of specific positions on the guanosine molecule and using defined analogs in competitive ELISA. RESULTS: Competitive inhibition assays revealed that serum antiguanosine antibodies bind across the 1 and 7 positions of the guanosine molecule (p < 0.01) and that an oxygen is necessary at position 6 in the molecule. 4H2 exhibited the same binding specificity for guanosine as human polyclonal antiguanosine antibodies, showing a conserved epitope across species. When the fine specificity was compared with known epitopes, the antiguanosine antibodies were found to have the internal image of a G-binding protein, identical to that of the Ha-ras oncogene product p21. CONCLUSION: The finding that antiguanosine autoantibodies vary directly with specific features of SLE, especially nephritis and polyserositis, suggests that they may contribute to the pathology of SLE. Our findings that antiguanosine antibodies have G-binding protein active site homology support the possibility that this species of antibody might interfere with cell signal transduction.

Abnormalities of serum antielastin antibodies in connective tissue diseases.

BACKGROUND: Antibodies (Abs) to alpha-elastin (elastin breakdown product) and tropoelastin (elastin precursor) are found in the serum of all human subjects and correlate with their respective serum peptide levels; however, peptide levels vary with age and some disease states. This study was undertaken to determine if serum elastin Abs, peptides, and elastin metabolism were altered in autoimmune diseases by detecting a changing ratio of serum anti-alpha:tropoelastin Ab levels. METHODS: Serum from patients with a variety of connective tissue diseases, including 28 with systemic lupus erythematosus (SLE), 24 with scleroderma, 18 with rheumatoid arthritis (RA), 10 with polymyositis, and 39 with vasculitis, was compared with serum from 19 age-matched healthy subjects for levels of antitropoelastin and anti-alpha-elastin Abs. RESULTS: We found an increase in IgG anti-alpha-elastin and a decrease in antitropoelastin Abs in the sera of patients with scleroderma (p < .02 and .00005) and SLE (p < .006 and .011). There was also a marked increase in anti-alpha-elastin Abs in patients with polyarteritis nodosa (p < .0005) and decreases in antitropoelastin Abs in patients with RA (p < .05), polymyositis (p < .01), and a variety of other vasculidities (p < .0003). CONCLUSIONS: Abnormal variations in elastin metabolism may be detected in several connective tissue diseases by measuring ratios of alpha- and tropoelastin IgG Abs as markers of elastin degradation and synthesis.

Immunohistological analysis of immune cell infiltration of a human colon tumor xenograft after treatment with Stealth liposome-encapsulated tumor necrosis factor-alpha and radiation.

The toxicity associated with tumor necrosis factor-alpha (TNF-alpha) has limited its usefulness as an anticancer agent. However, encapsulation of TNF-alpha in Stealth (SL) liposomes can minimize risk for toxicity and thus increase its potential as an adjuvant treatment. Our recent studies have shown that SL-TNF-alpha plus radiation is more effective at inhibiting LS174T colon tumor growth than either radiation alone or free TNF-alpha plus radiation. This increase in efficacy was coincident with a modulation of immune parameters in blood and spleen. The aim of this study was to determine if infiltration of natural killer (NK) cells, macrophages, and neutrophils into LS174T tumors was altered by SL-TNF-alpha treatment and whether any observed changes could potentially contribute to the enhanced antitumor efficacy seen with SL-TNF-alpha plus radiation treatment. Sections of excised tumors were examined histologically and quantitative analysis was performed using laser scanning cytometry. The data showed that the group receiving multiple treatments with SL-TNF-alpha plus radiation had the smallest tumors, but yet the level of necrosis was similar to that found in groups with much larger tumors. Furthermore, the necrotic areas in the SL-TNF-alpha plus radiation group had signs of recent and/or continuing cell death and the highest levels of NK cell and macrophage infiltrates. In time course experiments, a single injection of SL-TNF-alpha (but not free TNF-alpha) induced fluctuations in leukocyte infiltration into tumors that correlated inversely with our previous findings in blood and spleen. Overall, the data indicate that the mechanisms underlying the increased efficacy of SL-TNF-alpha compared to free TNF-alpha include a rapid and relatively sustained recruitment of NK cells, macrophages, and neutrophils.

Hypergravity-induced immunomodulation in a rodent model: lymphocytes and lymphoid organs.

The major goal of this study was to quantify changes in lymphoid organs and cells over time due to centrifugation-induced hypergravity. C57BL/6 mice were exposed to 1, 2 and 3 G and the following assays were performed on days 1, 4, 7, 10, and 21: spleen, thymus, lung, and liver masses; total leukocyte, lymphocyte, monocyte/macrophage, and granulocyte counts; level of splenocyte apoptosis; enumeration of CD3+ T, CD3+/CD4+ T helper, CD3+/CD8+ T cytotoxic, B220+ B, and NK1.1+ natural killer cells; and quantification of cells expressing CD25, CD69, and CD71 activation markers. The data show that increased gravity resulted in decreased body, spleen, thymus, and liver, but not lung, mass. Significant reductions were noted in all three major leukocyte populations (lymphocytes, granulocytes, monocyte/macrophages) [correction of macrphages] with increased gravity; persistent depletion was noted in blood but not spleen. Among the various lymphocyte populations, the CD3+/CD8+ T cells and B220+ B cells were the most affected and NK1.1+ NK cells the least affected. Overall, the changes were most evident during the first week, with a greater influence noted for cells in the spleen. A linear relationship was found between some of the measurements and the level of gravity, especially on day 4. These findings indicate that hypergravity profoundly alters leukocyte number and distribution in a mammalian model and that some aberrations persisted throughout the three weeks of the study. In certain cases, the detected changes were similar to those observed after whole-body irradiation. In future investigations we hope to combine hypergravity with low-dose rate irradiation and immune challenge.

Cytoskeletal and functional changes in bioreactor assembled thyroid tissue organoids exposed to gamma radiation.

Fischer rat thyroid cells were grown under low-shear stress in a bioreactor to a stage of organization composed of integrated follicles resembling small thyroid glands prior to exposure to 3 Gray-gamma radiation. Bioreactor tissues and controls (both irradiated and non-irradiated) were harvested at 24, 48, 96 and 144 hours post-exposure. Tissue samples were fixed and fluorescently labeled for actin and microtubules. Tissues were assessed for changes in cytoskeletal components induced by radiation and quantified by laser scanning cytometry. ELISA's were used to quantify transforming growth factor-beta and thyroxin released from cells to the culture supernatant. Tissue architecture was disrupted by exposure to radiation with the structural organization of actin and loss of follicular content the most obviously affected. With time post-irradiation the actin appeared disordered and the levels of fluorescence associated with filamentous-actin and microtubules cycled in the tissue analogs, but not in the flask-grown cultures. Active transforming growth factor-beta was higher in supernatants from the irradiated bioreactor tissue. Thyroxin release paralleled cell survival in the bioreactors and control cultures. Thus, the engineered tissue responses to radiation differed from those of conventional tissue culture making it a potentially better mimic of the in vivo situation.