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.

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.

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.

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.

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.