Sister chromatid exchanges in laboratory cultured cells after repeated exposures to pulsed ultrasound

In this in vitro study, suspensions of DON cells (lung diploid cell line from the Chinese hamster) were exposed to ultrasound regimes using pulse lengths similar to those used in ultrasonic Doppler measurements of fetal blood flow. The base level frequency of sister chromatid exchanges (SCE) was not significantly altered following exposure to a range of spatial peak temporal average intensities from 0.1 to 4.0 W/cm2. Repeating the exposure on three further occasions during the 20 generations following the initial insonation also had no effect. When DON cells were incubated in the presence of Mitomycin C (MCC), a positive dose-related increase in SCE frequency was observed, indicating the sensitivity of these cells to in vitro SCE induction. Coincidental exposure to ultrasound and Mitomycin C did not increase the SCE level above that obtained with MCC alone, and it was concluded that there was no evidence of a synergistic association.     

Sister chromatid exchanges in human lymphocytes after exposure to pulse-wave ultrasound

We have investigated the frequencies of sister chromatid exchange (SCE) induced by pulse-wave ultrasound in lymphocytes obtained from the human umbilical vein. The following results were obtained: There is an increase in the frequency of SCEs with increases in acoustic power. The critical acoustic power was 37.3-73 mW/cm2 (spatial average temporal average, SATA). Regardless of changes in pulse duration and repeated pulse frequency, if the acoustic power remains constant, the SCE frequency does not change significantly.     

Sister chromatid exchange analysis of human cells exposed to diagnostic levels of ultrasound.

Lymphocyte and lymphoblastoid cells were exposed in vitro to diagnostic levels of ultrasonic beams delivered by a Hewlett-Packard CE 30001 and a GE system with a 5 MHz linear transducer for 20 sec, 1 min, 5 min, and 20 min. Temperature and cavitation effects were controlled and there were matched sham exposures. The synergistic effects of theophylline with ultrasonography also were investigated. Small increases in sister chromatid exchange levels were observed after ultrasonic exposure, but increases were so small as to be unlikely to have clinical relevance. Theophylline was found to have no effect and ultrasonography had no effect on cell viability.     

Lack of effect of high-intensity pulsed ultrasound on sister chromatid exchange and in vitro Chinese hamster ovary cell viability

The frequency of sister chromatid exchanges (SCEs) in in vitro Chinese hamster ovary cells and their viability were not affected by 3-min exposures to 2-4 MHz, focused, pulsed ultrasound with a pulse repetition rate of 200 Hz, pulse duration of 10 mu sec and intensities (SPTP) of 500 W/cm2 and 2500 W/cm2. The viability results are consistent with those reported elsewhere; the SCE response does not verify a specific previously reported positive response.     

Sister chromatid exchange frequency in human lymphocytes after long duration exposure to pulsed ultrasound

The aim of this study is to determine whether sister chromatid exchange (SCE) frequency increases in human lymphocytes that are exposed to pulsed ultrasound conditions within the range typically employed in ultrasound Doppler measurements of fetal blood flow. Whole blood diluted with culture medium was exposed in vitro to 3.1 MHz pulsed ultrasound at spatial peak pulse average (SPPA) intensities from 15 to 135 W/cm2. Insonation for 5 min did not significantly alter the base level frequency of SCEs compared to that observed in sham-insonated controls. To avoid missing an effect, due to differing sensitivities of the division stages within the mitotic cycle, insonation was also applied for 24 hr to include one complete cell division cycle. No change in SCE was observed after this extended insonation at SPPA intensity of 33 W/cm2.     

Growth retardation in Chinese hamster V-79 cells exposed to 1 MHz ultrasound

Asynchronous Chinese hamster V-79 cells were exposed in suspension to continuous wave 1 MHz ultrasound for 1 min at an axial intensity of 2.5 W/cm², then allowed to grow in monolayer culture. There was little if any growth, as determined by changes in the number of attached cells, for 24 hr after sonication, then a return to normal growth approximately 36 hr after sonication. The lack of growth during the first 24 hr appears to be due to an approximate balance between proliferation of viable cells and loss of non-viable cells into the medium.     

Lack of induced increase in sister chromatid exchanges in human lymphocytes exposed to in vivo therapeutic ultrasound

In 1984 Stella et al. reported that each of 10 patients exposed to therapeutic ultrasound had a statistically significant increase in sister chromatid exchanges (SCEs) at mid- and end of therapy; there was a wide range of clinical symptoms (e.g., elbow epicondylitis to knee arthrosis) in this set of patients. In the present study, a set of 4 patients each with some diagnosis recommending therapeutic ultrasound was similarly tested for SCE induction; an additional set of 4 healthy persons underwent sham-therapeutic ultrasound exposures. For both sets of subjects (patients, nonpatients) there was no increase in the frequency of SCEs.     

The effects of diagnostic ultrasonography on the frequencies of sister chromatid exchanges in Chinese hamster cells and human lymphocytes

Human lymphocytes as well as Chinese hamster ovary cells were sonicated with either a fetal pulse detector (Siemens, Eucotone) or a compound scanner (Kretz, Combison 4100) at diagnostic energy levels. In one experimental series the cells were treated in the G1-phase of the cell cycle, in the other during late S-phase. The frequency of sister chromatid exchanges was determined in the metaphase chromosomes of the following mitosis. No significant increase in the frequency of SCEs per cell was observed as compared with the controls.     

Neurokinin(3) receptors couple to the activation of neuronal nitric-oxide synthase in stably transfected Chinese hamster ovary cells

Several physiological effects induced by activation of neurokinin(3) (NK(3)) receptors are mediated by the production of nitric oxide (NO). We investigated the intracellular coupling of NK(3) receptors to NO synthase (NOS) using a Chinese hamster ovary cell line that was stably transfected with both the NK(3) receptor and type I (neuronal) NOS. NOS activity in the transfected cell line was assayed directly, by measuring the formation of L-citrulline, another product of NOS, as well as indirectly, by measuring the production of cGMP in cultured rat fetal lung fibroblasts (RFL-6 cells). MePhe(7)-neurokinin B (NKB) stimulation of L-[(3)H]citrulline production was concentration-dependent and yielded a two-site model for the concentration-response relationship. The production of L-citrulline in response to two other tachykinins, substance P or neurokinin A, revealed only a one-site nature of the response. The production of cGMP in response to MePhe(7)-NKB had an EC(50) value that corresponded to the high-potency component of MePhe(7)-NKB-induced production of L-[(3)H]citrulline. Agonist-induced calcium signaling was also concentration-dependent, and the acute increase in the production of cGMP by MePhe(7)-NKB (0.1 nM) was dependent on the release of calcium from intracellular stores. Results of this study provide the first direct evidence that NK(3) receptors couple to the generation of NO within the same cell.     

Expression of vascular endothelial growth factor does not promote transformation but confers a growth advantage in vivo to Chinese hamster ovary cells.

Vascular endothelial growth factor (VEGF) is a mitogen with a specificity for endothelial cells in vitro and an angiogenic inducer in vivo. We tested the hypothesis that VEGF may confer on expressing cells a growth advantage in vivo. Dihydrofolatereductase--Chinese hamster ovary cells were transfected with expression vectors which direct the constitutive synthesis of VEGF. Neither the expression nor the exogenous administration of VEGF stimulated anchorage-dependent or anchorage-independent growth of Chinese hamster ovary cells in vitro. However, VEGF-expressing clones, unlike control cells, demonstrated an ability to proliferate in nude mice. Histologic examination revealed that the proliferative lesions were compact, well vascularized, and nonedematous. Ultrastructural analysis revealed that capillaries within the lesions were of the continuous type. These findings indicate that the expression of VEGF may confer on cells the ability to grow in vivo in the absence of transformation by purely paracrine mechanisms. Since VEGF is a widely distributed protein, this property may have relevance for a variety of physiological and pathological proliferative processes.     

高强度聚焦超声制备抗原致敏树突状细胞及其诱导细胞毒性T淋巴细胞杀伤效应的研究

目的 探讨高强度聚焦超声(HIFU)制备抗原致敏树突状细胞(DC)及其诱导细胞毒性T淋巴细胞(CTL)的杀伤效应.方法 应用小鼠重组GM-CSF和IL-4培养小鼠骨髓DC,用HIFU制备CT26肿瘤细胞抗原致敏DC疫苗,用3H-TdR法检测T细胞增殖反应的能力,标准的4 h 51Cr 释放测定法检测CTL杀伤活性.结果 HIFU组、肿瘤细胞冻融组、肿瘤上清组和PBS组的DC在体外均可以诱导CTL增殖,但HIFU组、肿瘤细胞冻融组的DC体外诱导CTL增殖能力明显高于肿瘤上清组和PBS组(P＜0.05).HIFU组和肿瘤细胞冻融组DC体外诱导的CTL对CT26结肠癌均有明显的细胞毒性作用,与肿瘤上清组DC和PBS组比较差异有统计学意义(P＜0.05),HIFU组体外诱导的CTL杀伤活性与肿瘤细胞冻融组比较差异无统计学意义(P＞0.05).结论 HIFU制备抗原致敏DC可以诱导CTL大量增殖,并能诱导出杀伤效应较强的CTL.     

Agonist-directed trafficking of porcine alpha(2A)-adrenergic receptor signaling in Chinese hamster ovary cells: l-isoproterenol selectively activates G(s)

In this study, we investigated the hypothesis of agonist-directed trafficking of receptor signaling for the alpha(2A)-adrenergic receptor (alpha(2A)-AR). alpha(2A)-ARs couple to both G(s) and G(i) to stimulate or inhibit adenylyl cyclase activity. Chinese hamster ovary-K1 cell lines expressing the porcine alpha(2A)-AR at high (alpha(2A)-H) and low (alpha(2A)-L) levels were used to estimate the relative efficacies (R.e.s) of a series of agonists for the G(s) and G(i) pathways. G(s)-mediated responses were measured after pertussis toxin treatment to inactivate G(i) in alpha(2A)-H, whereas G(i) responses were measured in alpha(2A)-L, where G(s) responses were absent. The full agonist UK-14,304 showed a large receptor reserve for G(i) responses in alpha(2A)-H but little receptor reserve for G(s) responses in alpha(2A)-H or for G(i) responses in alpha(2A)-L. With the exception of l-isoproterenol (ISO), all agonists showed similar R.e.s at the alpha(2A)-AR for G(s) and G(i) responses, with rank orders of R.e.s as follows: l-epinephrine = l-norepinephrine = UK-14,304 > p-aminoclonidine > or = BHT-920 > or = BHT-933 > clonidine = p-iodoclonidine > or = xylazine > or = guanabenz. Interestingly, ISO had the highest efficacy at the alpha(2A)-AR for activating G(s) versus G(i) (9-fold higher); however, it had low potency for both. By several criteria, the ISO response was mediated by the alpha(2A)-AR, supporting the hypothesis of agonist-directed trafficking of receptor signaling or agonist-specific G protein selectivity. In contrast, the apparent G(i) pathway selectivity of oxymetazoline appears to be mediated by an endogenous serotonergic receptor. It is intriguing that a classic beta-AR agonist that activates G(s) through beta(2)-ARs also appears to produce a G(s)-selective conformation of the G(i)-coupled alpha(2A)-AR.     

高强度聚焦超声制备树突状细胞瘤苗对BALB/c小鼠肿瘤主动免疫的实验研究

目的探讨采用高强度聚焦超声(HIFU)制备肿瘤抗原致敏树突状细胞(DC)对正常BALB/c小鼠肿瘤的主动免疫作用。方法采用功率1000W/cm2的HIFU辐照CT-26结肠癌细胞30s,获取肿瘤抗原,与体外培养扩增第5天的DC共孵育,制备DC瘤苗。HIFU辐照法制备的DC瘤苗(HIFU组)一次性免疫正常BALB/c小鼠,7d后再皮下接种活的CT-26结肠癌细胞,然后观察肿瘤发生时间、20d时瘤体重量和体积、小鼠生存时间等。并设立反复冻融法致敏DC组(冻融组)和生理盐水对照组(对照组),比较各组瘤体重量和体积、小鼠生存时间是否有差异。结果HIFU组、冻融组与对照组比较肿瘤发生时间、20d瘤体重量和体积、小鼠中位生存时间等差异均有统计学意义(P<0.01或P<0.05)。HIFU组与冻融组比较瘤体重量、体积及小鼠中位生存时间差异亦有统计学意义(P<0.05)。结论HIFU辐照法制备的DC瘤苗对小鼠肿瘤的发生有一定的主动免疫作用,并优于反复冻融法制备的DC瘤苗。     

脉冲高强度聚集超声增强裸质粒在鳞癌模型中转染与表达的初步研究

目的：探讨脉冲高强度聚焦超声局部辐照荷瘤裸鼠后。对经尾静脉输入的报告基因在瘤体中转染和表达的影响。材料与方法：于雌性C3H裸鼠（n=3）两侧腋窝皮下接种鳞癌组织块,待肿瘤长到体积约0．4cm^3时进行实验。每只裸鼠一侧肿瘤采用脉冲高强度聚集超声辐照,另一侧作为对照。构建报告基因表达载体,超声辐照后,立即由尾静脉输入。24h后处死动物,在荧光显微镜下观察组织切片中绿色荧光蛋白（GFP）的表达;Westren Blot免疫印迹技术定量分析GFP信号强度;进行组织特异性染色观察超声辐照后肿瘤细胞受损情况（苏木精—伊红染色,末端脱氧核苷酸转移介导脱氧尿苷三磷酸切口末端标记）。     

Increased expression of vascular cell adhesion molecule-1 (VCAM-1) in cultured endothelial cells exposed to serum from type 1 diabetic patients: no effects of high glucose concentrations

Activation of the arterial endothelium may play an important role in the development of an atherosclerosis-prone vascular wall in diabetes. The induction of the adhesion molecules VCAM-1 and E-selectin on activated endothelial cells is crucial in monocyte recruitment during the atherogenic process. In the present study, we investigated whether sera from type 1 diabetic patients and non-diabetic persons are capable of inducing expression of VCAM-1 and E-selectin in human endothelial cells cultured in vitro. First, it was found that the addition of serum from non-diabetics to the cultures resulted in expression of adhesion molecules above basal level and also increased the cellular response to the cytokine tumor necrosis factor-alpha (TNF-alpha), a strong inducer of both adhesion molecules. Moreover, it was found that, on average, sera from 17 diabetic males induced a higher expression of VCAM-1 in the endothelial cells after 6 h of incubation than samples from 20 non-diabetic age-matched males (p < 0.05). No difference between the diabetic and non-diabetic group was seen in the expression of E-selectin. Likewise, no differences were observed between the effects of the sera to induce TNF-alpha responsivity. A series of experiments showed that alterations in the glucose concentrations of the growth medium (5.5-13.5 mmol/L) did not change the cellular content of either VCAM-1 or E-selectin before and after TNF-alpha treatment. In conclusion, it has been shown that sera from diabetic patients contain component(s), capable of inducing VCAM-1 expression in endothelial cells independent of hyperglycemia. Augmented induction of endothelial VCAM-1 expression by circulating factor(s) may play a role in the development of atherosclerosis in diabetes.     

Increased expression of vascular cell adhesion molecule-1 (VCAM-1) in cultured endothelial cells exposed to serum from type 1 diabetic patients: no effects of high glucose concentrations

Activation of the arterial endothelium may play an important role in the development of an atherosclerosis-prone vascular wall in diabetes. The induction of the adhesion molecules VCAM-1 and E-selectin on activated endothelial cells is crucial in monocyte recruitment during the atherogenic process. In the present study, we investigated whether sera from type 1 diabetic patients and non-diabetic persons are capable of inducing expression of VCAM-1 and E-selectin in human endothelial cells cultured in vitro . First, it was found that the addition of serum from non-diabetics to the cultures resulted in expression of adhesion molecules above basal level and also increased the cellular response to the cytokine tumor necrosis factor-alpha (TNF- &#102 ), a strong inducer of both adhesion molecules. Moreover, it was found that, on average, sera from 17 diabetic males induced a higher expression of VCAM-1 in the endothelial cells after 6 h of incubation than samples from 20 non-diabetic age-matched males (p<0.05). No difference between the diabetic and non-diabetic group was seen in the expression of E-selectin. Likewise, no differences were observed between the effects of the sera to induce TNF- &#102 responsivity. A series of experiments showed that alterations in the glucose concentrations of the growth medium (5.5 - 13.5 mmol/L) did not change the cellular content of either VCAM-1 or E-selectin before and after TNF- &#102 treatment. In conclusion, it has been shown that sera from diabetic patients contain component(s), capable of inducing VCAM-1 expression in endothelial cells independent of hyperglycemia. Augmented induction of endothelial VCAM-1 expression by circulating factor(s) may play a role in the development of atherosclerosis in diabetes.