Effect of low dose rate irradiation on the division potential of cells in vitro. IV. Embryonic and adult human lung fibroblast-like cells

Early and late passage human embryonic lung fibroblasts were compared with early passage adult lung fibroblasts with regards to their survival (number of population doublings), after low dose rate ionizing radiation. It was found that early passage embryonic cells are quite resistant to this type of radiation. Late passage embryonic and early passage adult fibroblasts are more sensitive to ionizing radiations. The results suggest that cell aging is accompanied by an increased sensitivity to low dose rate ionizing radiation and favor the idea that aging in vitro, expressed as a function of the fibroblast division potential, is correlated with aging in vivo.     

Induction of chromosome aberrations in human lymphocytes by cyclophosphamide action in vivo and in vitro

The dose dependences of chromosome aberrations rate were investigated in lymphocytes of oncological patients after cyclophosphamide (CP) administration. It was shown that the rate of chromosome aberrations and the number of disruptions per cell in vivo and in vitro increases exponentially with the dose. At the same time, the parameters of regression equations coincide. This evidences that CP has the similar effect in vivo and in vitro.     

Perturbation of cell division by acrylamide in vitro and in vivo

Exposure of V79 Chinese hamster cells to acrylamide (AA) caused a concentration-dependent increase in the incidence of spindle disturbances. A c-mitotic effect with the appearance of C-metaphases, a mitotic block and the concomitant disappearance of ana-telophase figures, was observed after 6 h of treatment with concentrations ranging from 0.01 to 1.0 mg/ml of AA.Intraperitoneal injection of male mice with the highest tolerated dose of 120 mg/kg of AA showed no mitotic arrest in bone marrow cells. However, 1 h and 3 h after treatment the frequencies of cells with highly condensed and separated chromatids was reduced indicating an effect on mitotic progression.In spermatocytes of mice AA caused a meiotic delay from 2 h to 22 h after treatment determined by a reduced ratio of second/first meiotic divisions. The meiotic delay was predominantly due to a prolongation of interkinesis.The present results show that AA causes disturbances of cell division in vitro and in vivo. They suggest that AA might induce aneuploidy in mammalian cells in vitro by interfering with proper functioning of the spindle similar to the effect of colchicine. In vivo, particularly in spermatocytes, the progression of cell division was altered by AA. It cannot be explained simply by an effect on spindle function however, this alteration may also cause errors in chromosome segregation.     

The effect of ethylnitrosourea on chromosome aberrations in vitro and in vivo.

The effect of ENU on (A) human chromosomes from blood lymphocyte cultures in vitro, and on (B) rat and mouse bone marrow chromosomes in vivo, was investigated. Doses of 25, 50, 100 and 200 mug/ml were tested in vitro and cells with chromosome breakage were found to be dose dependent. Chromosome damage was also dependent on time; maximum damage was seen when cells were treated 2--6 hrs before harvest. Two doses of 100 and 200 mg/kg were studied in rat and mouse in vivo and a dose effect could be shown in both species. The highest number of abnormal cells was found 6 hrs after treatment; there was a sharp decrease at 18 hrs and thereafer. Types of aberrations were also analyzed, in both in vitro and in vivo studies.     

In vitro and in vivo expression of endothelial von Willebrand factor and leukocyte accumulation after fractionated irradiation

Previous investigations have demonstrated an increased release of von Willebrand factor (VWF; also known as vWF) in endothelial cells after high single-dose irradiation in vitro. We have also found increased levels of Vwf protein in mouse glomeruli after a high single dose of renal irradiation in vivo. In addition, increased numbers of leukocytes were observed in the renal cortex after irradiation in vivo. The aim of the present study was to investigate and quantify these biological processes after clinically relevant fractionated irradiation and to relate them to changes in renal function. A significantly greater increase in release of VWF was observed in cultured human umbilical vein endothelial cells (HUVECs) after fractionated irradiation (20 x 1.0 Gy) than after a single dose of 20 Gy (147% compared to 115% of control, respectively, P < 0.0005). In contrast with the in vitro observations, glomerular Vwf staining was lower after fractionated irradiation in vivo (20 x 2.0 Gy or 10 x 1.6 Gy +/- re-irradiation) than after a single dose of 16 Gy. The number of leukocytes accumulating in the renal cortex was also lower after fractionated in vivo irradiation than after a single radiation dose. The onset of these events preceded renal functional and histopathological changes by approximately 10 weeks. These data indicate that radiation-induced changes in endothelial VWF expression after in vivo irradiation may be distinct from the in vitro observations. Increased VWF expression may reflect pivotal processes in the pathogenesis of late radiation nephropathy and provide a clue to appropriate timing of pharmacological intervention.     

The cytokinesis-block micronucleus assay as a biological dosimeter in spleen and peripheral blood lymphocytes of the mouse following acute whole-body irradiation

To evaluate the application of the cytokinesis-block (CB) micronucleus (MN) assay as a biological dosimeter following in vivo exposure to ionising radiation we determined the micronucleus frequency in spleen and peripheral blood lymphocytes of the mouse, serially, for 14 days following acute whole-body irradiation. The baseline MN frequency of spleen lymphocytes (7.86 +/- 0.68, mean +/- 1 SD) was significantly (p less than 0.001) elevated when compared to that for peripheral blood lymphocytes (4.10 +/- 0.53). Immediately after irradiation there was a substantial dose-related increase in MN, but the MN frequencies in spleen lymphocytes (120.2 +/- 9.4 for 1 Gy; 409.5 +/- 38.4 for 2 Gy) were significantly (p less than 0.009) elevated compared to those in peripheral blood lymphocytes (78.0 +/- 7.0 for 1 Gy; 200.2 +/- 10.9 for 2 Gy). During the 14 days after irradiation, the MN frequency in spleen lymphocytes declined gradually to approximately half of the value observed immediately after irradiation. By contrast the MN frequency in peripheral blood lymphocytes increased during the week after irradiation, but ultimately MN frequencies in blood and spleen became approximately the same by day 14. Study of isolated murine lymphocytes irradiated in vitro showed that the number of MN generated by a given dose of radiation was approximately 2-3 times greater than the number generated by in vivo irradiation. These results suggest that measurement of MN in vivo after irradiation can be used as an in vivo dosimeter. However, precise dosimetry is probably affected by factors such as kinetic changes in different lymphocyte populations and possibly by in vivo factors which influence sensitivity of cells to radiation.     

The participation of oxygen in the process of the interphase death of T-lymphocytes following irradiation in vivo

The ability of lymphocytes to inhibit proliferation of non-syngeneic stem cells decreases differently after exposure in vivo and in vitro. The causes of the observed differences and the mechanism of radiation impairment of this function under different irradiation conditions have been investigated. Cells exposed in vivo die in the interphase irreversibly. The newly formed lymphocytes start the repair process as late as one month after irradiation. The injury to in vivo exposed cells is severer due to the presence of oxygen in tissues. A definite time interval is needed for the damaging effect of oxygen radicals to be implemented: the effect is maximum as early as 4 h following irradiation. With in vivo exposure under hypoxic conditions the functional activity of lymphocytes is the same as that of lymphocytes irradiated in vitro with the same dose. In vitro irradiation of lymphocytes at a high oxygen content causes a decrease in the functional activity of cells.     

Multiple mechanisms of B cell immunoregulation in man after administration of in vivo corticosteroids

Despite the extensive clinical use of corticosteroids (CS) in the treatment of immune-mediated diseases, relatively little is known concerning the effects of CS on B cell function as measured by in vitro assays. The effects of single-dose vs several days of in vivo CS therapy on the spontaneous and mitogen-induced Ig production by human peripheral blood B cells are reported here. Spontaneous Ig production by individual B cells was enhanced by in vivo CS as measured by an in vitro plaque-forming cell (PFC) assay. The same increased response was also observed with a brief in vitro exposure of the B cells to CS, which suggests that a mere brief exposure to an active CS analogue is all that is required to produce the enhanced response. The immunoregulatory effects of in vivo CS on mitogen-induced Ig production are more complex. Pokeweed mitogen-induced PFC responses were suppressed 4 to 5 hr after a single in vivo pharmacologic dose of CS, with complete recovery by 24 hr. In contrast, after a 5-day course of CS, the suppressed PFC response did not recover until 60 hr after the last dose. Moreover, several mechanisms of suppression were operative. Ten hours after completing the 5-day course of CS, there was a relative enrichment in the peripheral blood compartment of lymphocytes bearing the OKT8 suppressor/cytotoxic T cell phenotype that coincided with a depressed PFC response. At 36 hr after the last dose, the T lymphocyte profile returned to normal while B cell function remained suppressed. The complex, multifaceted modulation of the immune response, resulting from redistribution of cell subsets as well as altered cell functions, vary with time-dose parameters of in vivo CS administration. These observations should provide additional insights into the heterogeneity of CS-induced therapeutic effects.     

Glial repair in the cultured central nervous system of an insect

Summary Insect glial cells are capable of division and repair in organ culture after selective damage with the toxin ethidium bromide. The repair is slower and less organised than seen in vivo after similar treatment and is still incomplete after one month. Granule-containing cells, which play an important role in the early stages of repair in vivo, are never seen in cultured connectives. This observation adds further support to the hypothesis that these cells are derived from haemocytes and that their presence is necessary for rapid and orderly repair. The uptake of ³H-thymidine into perineurial glial cells in vitro, both in control and ethidiumtreated connectives, shows that there is a considerable proliferation of cells in this region. Some uptake of thymidine is also seen in subperineurial glia but division alone cannot account for the large increase in the number of glial nuclei found at the early stages of repair in this region. Further, glial cells with diverse morphologies suggest that subpopulations are present. We conclude that cell migration from undamaged areas, as well as cell proliferation, is necessary for CNS repair in vitro.     

IL-2 regulates expansion of CD4+ T cell populations by affecting cell death: insights from modeling CFSE data

It is generally accepted that IL-2 influences the dynamics of populations of T cells in vitro and in vivo. However, which parameters for cell division and/or death are affected by IL-2 is not well understood. To get better insights into the potential ways of how IL-2 may influence the population dynamics of T cells, we analyze data on the dynamics of CFSE-labeled polyclonal CD4(+) T lymphocytes in vitro after anti-CD3 stimulation at different concentrations of exogenous IL-2. Inferring cell division and death rates from CFSE-delabeling experiments is not straightforward and requires the use of mathematical models. We find that to adequately describe the dynamics of T cells at low concentrations of exogenous IL-2, the death rate of divided cells has to increase with the number of divisions cells have undergone. IL-2 hardly affects the average interdivision time. At low IL-2 concentrations 1) fewer cells are recruited into the response and successfully complete their first division; 2) the stochasticity of cell division is increased; and 3) the rate, at which the death rate increases with the division number, increases. Summarizing, our mathematical reinterpretation suggests that the main effect of IL-2 on the in vitro dynamics of naive CD4(+) T cells occurs by affecting the rate of cell death and not by changing the rate of cell division.     

地参多糖对实验肿瘤细胞体内外增殖的影响

旨在研究地参多糖对小鼠体内S180A肿瘤细胞增殖的影响及体外对人肝癌BEL-7402细胞生长的影响.体内实验采用皮下接种小鼠S180A肿瘤细胞,以生理盐水为阴性对照,环磷酰胺为阳性对照,腹腔给药(ip给药)不同剂量地参多糖7d,称取小鼠瘤质量并计算抑瘤率.体外实验采用四甲基偶氮唑盐(MTT)法测定地参多糖对体外BEL-7402细胞抗肿瘤活性;倒置荧光显微镜观察地参多糖对BEL-7402细胞形态学的影响;流式细胞术检测地参多糖对人肝癌BEL-7402细胞周期的影响.体内实验结果表明:随着地参多糖浓度的增加,体内对小鼠S180A肿瘤的抑制率逐渐提高,最高抑瘤率可达41.29％ (P＜0.01).体外实验结果表明:随着地参多糖浓度和培养时间的增加,体外培养的BEL-7402细胞存活率逐渐降低,抑制率逐渐增加,细胞出现明显的形态学改变,且能诱导细胞凋亡;流式细胞术证实地参多糖使体外培养的BEL-7402细胞发生G0/G1期阻滞.体内外实验均证实地参多糖具有抗肿瘤作用,值得进一步开发利用.     

The preimplantation pig embryo: cell number and allocation to trophectoderm and inner cell mass of the blastocyst in vivo and in vitro

Total cell number as well as differential cell numbers representing the inner cell mass (ICM) and trophectoderm were determined by a differential staining technique for preimplantation pig embryos recovered between 5 and 8 days after the onset of oestrus. Total cell number increased rapidly over this time span and significant effects were found between embryos of the same chronological age from different females. Inner cells could be detected in some but not all embryos of 12-16 cells. The proportion of inner cells was low in morulae but increased during differentiation of ICM and trophectoderm in early blastocysts. The proportion of ICM cells then decreased as blastocysts expanded and hatched. Some embryos were cultured in vitro and others were transferred to the oviducts of immature mice as a surrogate in vivo environment and assessed for morphology and cell number after several days. Although total cell number did not reach in vivo levels, morphological development and cell number increase was sustained better in the immature mice than in vitro. The proportion of ICM cells in blastocysts formed in vitro was in the normal range.     

Ah locus-associated differences in induction of sister-chromatid exchanges and in DNA adducts by benzo[a]pyrene in mice.

The effect of alleles of the Ah locus on the induction of sister-chromatid exchanges (SCE) was studied in C57Bl/6 and in DBA/2 mice treated twice intragastrically with benzo[a]pyrene (BP, 100 or 10 mg/kg b.w.). To measure the changes in the frequency of SCE, 2 protocols were used: in vivo in bone marrow cells after implantation of 5-bromodeoxyuridine (BrdU) tablets and in vivo/in vitro in spleen lymphocytes cultured with BrdU. On day 5 mice were killed and SCEs estimated in bone marrow cells. BP-DNA adducts in bone marrow and spleen were analyzed on day 5 after the same exposure to BP. In the spleen lymphocytes SCE frequencies were analyzed after an additional 48 h of culture. We found that at both doses of BP, the number of SCEs and BP-DNA adducts in bone marrow and in spleen cells was significantly higher in aryl hydrocarbon hydroxylase (AHH)-non-inducible (DBA/2) mice than in AHH-inducible (C57BL/6) mice. Only marginal induction of SCE was noted after the high dose of BP in C57BL/6 mice in bone marrow in vivo, whereas a highly significant increase in the frequency of SCEs was found in splenocytes in the in vivo/in vitro test. The spleen cells contained larger amounts of BP-DNA adducts and demonstrated higher absolute levels of SCEs than bone marrow cells. The sensitivity of both the in vivo/in vitro and the in vivo SCE test is high enough for assessment of Ah locus-linked differences in BP genotoxicity in mice at the prolonged time between treatment and cell preparation. The present data confirm the influence of inducibility of AHH in the intestine on the genotoxicity of BP to distal tissues after oral exposure to BP.     

Measurement of micronuclei in lymphocytes

The micronucleus technique has been proposed as a method for measurement of chromosomal damage in mitogen-stimulated human lymphocytes. Micronuclei require one cell division to be expressed and, consequently, the conventional micronucleus technique is very imprecise since the cells which have undergone only one division, and the micronuclei in them, cannot be identified separately from the total population of lymphocytes. To overcome this problem, two methods were developed to identify cells which have undergone their first mitosis. Using an autoradiographic technique, lymphocytes were pulse-labelled with [3H]thymidine at 48 h of culture, allowed to proceed through mitosis, identified by autoradiography between 72 and 84 h and micronuclei were scored in them. It was not possible to select a concentration of radiolabel which did not itself produce micronuclei and consequently the method was of no value for measuring pre-existing chromosomal damage present in vivo. However, it was capable of quantitating micronuclei produced by irradiation of lymphocytes in vitro. In the second method, cytokinesis was blocked using cytochalasin B. Micronuclei were scored in cytokinesis-blocked cells. These were easily recognisable owing to their binucleate appearance and a large number could be accumulated by adding 3.0 micrograms/ml cytochalasin B at 44 h and scoring at 72 h. Cytochalasin B did not itself produce micronuclei. The cytokinesis-block method was simple to perform; the 'in vivo' micronucleus frequency in normal individuals was 4.4 +/- 2.6 micronuclei/500 cytokinesis-blocked cells; and for lymphocytes irradiated in vitro there was a linear relationship between dose of radiation and number of induced micronuclei. The cytokinesis-block method appears to be the procedure of choice for quantitating micronuclei in lymphocytes.     

The effect of interleukin-17 on hematopoietic cells and cytokine release in mouse spleen

To evaluate whether the response of hematopoietic cells to interleukin-17 (IL-17) depends on the tissue microenvironment in which hematopoiesis occurs, the influence of recombinant mouse IL-17 on spleen hematopoietic cells and cytokine release was assessed in normal mice in vitro and in vivo. In vitro, IL-17 did not significantly affect the growth of granulocyte-macrophage (CFU-GM) and erythroid (BFU-E and CFU-E) derived colonies. A single injection of IL-17 in vivo exhibited stimulatory effects on hematopoietic cells from both granulocytic and erythroid lineages. The increased number of metamyelocytes 48 h after treatment imply to the IL-17-induced stimulation of granulopoiesis. The number of BFU-E was increased at 24 h, while the number of CFU-E increased 6 h and 24 h after treatment. Since the same treatment in the bone marrow decreased the number of CFU-E, it may be concluded that the local microenvironment plays an important role in IL-17-mediated effects on CFU-E. IL-17 increased the release of IL-6 both in vitro and in vivo, but showed tendency to suppress the constitutive secretion of IL-10 by spleen cells. Our results suggest the complexity of target cell response and interplay of secondary induced cytokines by IL-17 in different hematopoietic organs.     

Testosterone metabolism in neuroendocrine organs in male rats under atrazine and deethylatrazine influence

The inhibitory influence of atrazine and deethylatrazine on testosterone metabolism in male rat anterior pituitary and hypothalamus were studied under in vivo and in vitro experimental conditions. In vivo strong influence of atrazine (12 mg/100 g by wt. daily during 7 days) on 5 alpha-R, 3 alpha- and 17 beta-HSD activities was detected in the anterior pituitary. This dose provokes a significant increase in the weight of the pituitary gland, with hyperemia and hypertrophy of chromophobic cells with vacuolar degeneration. In vivo treatment of male rats with the same dose of deethylatrazine markedly inhibited 5 alpha-R activity in the anterior pituitary. The rate of 5 alpha-R activity inhibition in the anterior pituitary was the same after in vivo treatment with atrazine (37.3%) as with deethylatrazine (33.9%). This could suggest that the mechanism of inhibition of deethylatrazine is similar to that of atrazine. In vitro atrazine or deethylatrazine addition into the incubation medium significantly (P less than 0.01) inhibited 5 alpha-R, 3 alpha- and 17 beta-HSD activities in the anterior pituitary. The inhibition of 5 alpha-R activity was marked more by atrazine than deethylatrazine, while 3 alpha- and 17 beta-HSD activities were inhibited at the same rate. In vivo treatment with the same dose of atrazine or deethylatrazine (12 mg/100 g by wt daily 7 days) significantly inhibited (P less than 0.01) 5 alpha-R and 17 beta-HSD at the male rat hypothalamic level. 3 alpha-HSD activity inhibition was not significant for either compound. The in vitro addition of deethylatrazine was much more effective (P less than 0.01) in inhibiting 5 alpha-R, 3 alpha- and 17 beta-HSD in male rat hypothalamus than atrazine. In spite of this, deethylatrazine seems to be less toxic in in vivo experiments due to its higher polarity and faster biodegradation.     

Biological consequences of irradiation by low doses of technetium 99m: ultrastructural studies, p53 protein expression and cytogenetic effects.

Few studies concerning the potential genetic effects of diagnostic radionuclides used in nuclear medicine have been reported. The aim of this study was to evaluate the biological and cytogenetic consequences of two technetium 99m-labelled radiopharmaceuticals. Ultrastructural modifications of pulmonary cells were first investigated after injection of 99mTc labelled microspheres in the rat. On the same irradiated cells, nuclear expression of p53 protein was assessed using immunohistochemistry. Despite very high previously calculated doses delivered to pulmonary cells, no morpholological cell damage and no significant increase of nuclear expression of the p53 were noted. There was no correlation between the calculated dose and the ultrastructural biological damage. Secondly, a specific in vitro curve, activity/number of unstable chromosomal aberrations, corresponding to physical characteristics of 99mTc, was established to verify the potentiality of 99mTc to induce such aberrations. In vivo, cytogenetic effects were assessed on blood samples of 5 patients with various arthrosic and periarthrosic diseases obtained after bone scintigraphy. Aberration frequencies of both in vitro and in vivo irradiated lymphocytes were determined using the classical Fluorescence Plus Giemsa technique. No cytogenetic effects appeared with the routinely 99mTc injected activities as predicted by the in vitro curve.     

Disparate in vitro and in vivo requirements for IL-2 during antigen-independent CD8 T cell expansion

Transient TCR stimulation induces multiple rounds of CD8 T cell division without further requirement for Ag. The mechanism driving Ag-independent proliferation, however, remains unclear. In this study, we show that the initial duration of TCR stimulation positively correlates with the number of divisions that CD8 T cells subsequently undergo. We find that increased periods of Ag stimulation result in enhanced CD25 up-regulation and greater IL-2 production by CD8 T cells. Depletion of IL-2 from T cell cultures with specific Abs dramatically impairs programmed proliferation. Consistent with this result, IL-2-deficient T cells undergo markedly attenuated Ag-independent proliferation in vitro. Although IL-2 production by stimulated CD8 T cells appears to be essential for in vitro proliferation, upon transfer into recipient mice, IL-2-deficient CD8 T cells undergo extensive proliferation in vivo after transient stimulation. Furthermore, the extent of in vivo proliferation correlates with the duration of in vitro Ag stimulation. These results indicate that the requirements for autocrine IL-2 production by CD8 T cells differs between in vitro and in vivo conditions and suggests that factors in addition to IL-2 can support Ag-independent CD8 T cell proliferation.     

Comparison of the frequency of chromosome aberrations induced by thiophosphamide in rabbit lymphocytes in vitro and in vivo

Rabbit lymphocytes were treated with thiophosphamide in vivo and in vitro. In-vitro doses were calculated by multiplying thiophosphamide concentrations by the time of treatment (the doses ranged within 0-1500 mg/min/ml). In-vivo doses were calculated as integral of thiophosphamide concentration function from the time of administration till the time of blood sample collection (the doses ranged within 0-1900 mg/min/ml). It was shown that with the dose increase the rate of chromosome aberrations and the number of disruptions per cell rise exponentially in vivo and in vitro. At the same time the parameters of regression equations coincide. This evidences that thiophosphamide produces the same effect in vivo and in vitro.     

T cell activation: in vivo veritas

Phenotypic changes in CD4(+) T cells undergoing antigen-dependent activation were compared in vivo and in vitro. The most obvious difference was in expression of CD25, the alpha chain of the high affinity receptor for IL-2. High level expression of CD25 in vivo is restricted to a small fraction of the cells at the leading edge of the cell division profile, whereas all activated cells express high levels of CD25 in cultures responding to antigen. Because IL-2 is known to upregulate expression of CD25 in preactivated T cells, this suggests a difference in IL-2 exposure in the two responses. A number of other markers, including CD54, show a similar difference in the pattern of expression in vivo and in vitro. Using 6-colour flow cytometry, it was demonstrated that the small percentage of cells expressing CD25 in vivo coexpresses a very high level of a number of other activation markers, including CD38, CD44 and Ly-6A/E, suggesting that these may also be upregulated by autocrine IL-2.