Noninvasive assessment and quantification of tumour vascularisation using MRI and CT in a tumour model with modifiable angiogenesis - An animal experimental prospective cohort study.

BACKGROUND: To investigate vascular-related pathophysiological characteristics of two human lung cancers with modifiable vascularisation using MRI and CT. METHODS: Tumour xenografts with modifiable vascularisation were established in 71 rats (approval by the Animal Care Committee was obtained) by subcutaneous transplantation of two human non-small-cell lung cancer (NSCLC) cells (A549, H1299) either alone or co-transplanted with vascular growth promoters. The vascularity of the tumours was assessed noninvasively by MRI diffusion-weighted-imaging (DWI), T2-weighted, and time-of-flight (TOF) sequences) as well as contrast-enhanced CT (CE-CT), using clinical scanners. As a reference standard, histological examinations (CD-31, fluorescent beads) were done after explantation. RESULTS: Microvessel density (MVD) was higher in co-transplanted tumours (171 ± 19 number/mm2) than in non-co-transplanted tumours (111 ± 11 number/mm2; p = 0.002). Co-transplanted tumours showed higher growth rates and larger tumour vessels at TOF-MRI as well as larger necrotic areas at CE-CT. In co-transplanted tumours, DWI revealed higher cellularity (lower minimal ADCdiff 166 ± 15 versus 346 ± 27 mm2/s × 10-6; p < 0.001), highly necrotic areas (higher maximal ADCdiff 1695 ± 65 versus 1320 ± 59 mm2/s × 10-6; p < 0.001), and better-perfused tumour stroma (higher ADCperf 723 ± 36 versus 636 ± 51 mm2/s × 10-6; p = 0.005). Significant correlations were found using qualitative and quantitative parameters: maximal ADCperf and MVD (r = 0.326); maximal ADCdiff and relative necrotic volume on CE-CT (r = 0.551); minimal ADCdiff and MVD (r = -0.395). CONCLUSIONS: Pathophysiological differences related to vascular supply in two human lung cancer cell lines with modifiable vascularity are quantifiable with clinical imaging techniques. Imaging parameters of vascularisation correlated with the results of histology. DWI was able to characterise both the extent of necrosis and the level of perfusion.

Radiation-induced cell cycle arrests in Ehrlich ascites carcinoma cells in vivo.

Radiation-induced progression delay in G(1)/S, S and G(2)/M phases of p53 wild-type Ehrlich ascites carcinoma (EAC) cells growing in vivo was investigated by DNA flow cytometry. Different behavior patterns of EAC cells at the time after irradiation with low (2, 4, 6, 8 Gy) and high (10, 15, 20 Gy) doses were evaluated. While EAC cells showed a small progression delay in S phase and a dose-dependent block in G(2)/M phase after the irradiation with low doses, a clear additional block in G(1)/S phase was observed after irradiation with high doses. An assessment of the damage response and repair networks at the time after irradiation might have important implication for the development of cancer management and treatment.

Tumour bed irradiation of human tumour xenografts in a nude rat model using a common X-ray tube.

Studies that investigate the radiation of human tumour xenografts require an appropriate radiation source and highly standardized conditions during radiation. This work reports on the design of a standardized irradiation device using a commercially available X-ray tube with a custom constructed lead collimator with two circular apertures and an animal bed plate, permitting synchronous irradiation of two animals. Dosimetry and the corresponding methodology for radiotherapy of human non-small cell lung cancer xenograft tumours transplanted to and growing subcutaneously on the right lower limb in a nude rat model were investigated. Procedures and results described herein prove the feasibility of use of the device, which is applicable for any investigation involving irradiation of non-tumorous and tumorous lesions in small animals.

Distribution of fluorescent microspheres in vascular space and parenchymal organs of intact nude rats.

PURPOSE: To assess kinetics of elimination of different sized microspheres (MS) from the blood pool and tendency of their distribution in parenchymal organs of intact nude rats. MATERIALS AND METHODS: A mixture of 1 microm and 3 microm MS in phosphate-buffered saline was injected intravenously into eight rats under intraperitoneal anaesthesia. Blood samples were collected before, just after and in 2, 5 and 10 min after MS injection. Dynamics of MS elimination from blood pool was evaluated with flow cytometry. After euthanasia, histological sections were prepared and distributions of MS through the liver, spleen, kidney and lung were analysed with fluorescence microscopy and flow cytometry. RESULTS: The number of microspheres registered in the intravascular space showed a marked exponential decrease over time independent of MS size. Different amounts and proportions of 1 microm and 3 microm MS were revealed in lung, liver, spleen and kidneys of the rats. Most of 1 microm MS were localised in liver and spleen. In contrast, 3 microm MS were detected predominantly in lung. CONCLUSION: 1 microm and 3 microm MS may be assumed as free circulating particles only for a short period of time after injection. Their elimination kinetics seems to be tightly linked to specific tissue properties such a pulmonary vasoconstriction and phagocytosis.

[Main milestones of development and application of ductal cytometry at the Russian Federal Research Center for Radiology and Surgical Technology, St. Petersburg].

Ductal cytometry provides data on cellular DNA and RNA levels and overall profile of specific proteins identifiable by monoclonal antibodies. Results of its long-term use in clinical and oncological research are presented. Application of dosage ranging 0.28-1.1 mGy/sec was followed by stable 1.8-2-fold increase in the myelokariocyte profile cbering DNA synthesis. Bone marrow proliferation did not increase until relatively low dosage was used. A study of combined effects of prolonged gamma irradiation and lead and cadmium ions on rat's hemopoiesis pointed to radiation as the sole causative factor when cadmium chloride was used. Hemopoietic characteristics came back to normal when a combination of lead acetate and ionizing radiation was used, as a result of the oppositely directed action of the two factors. Standard monoclonal antibodies should not be employed for evaluating immunological vigor of patients with malignant gliomas due to the presence of a specific pathological link in their immune system.

Retrospective analysis of radiation load during examination of cancer patients by (68)Ga-DOTATOC and (18)F-FDG positron emission tomography.

We present retrospective analysis of the results of examinations of 1,338 cancer patients by (68)Ga-DOTATOC and (18)F-FDG positron emission and computer-aided tomography. It was shown that complex devices for positron emission and computer-aided tomography provide more informative data than individual methods. The protocol for examination by methods of positron emission and computer-aided tomography in each case is determined by clinical requirements and risk of extra exposure of the patient.

Varying responses of human cells with discrepant p53 activity to ionizing radiation and heat shock exposure.

OBJECTIVES: Both heat shock (HS) and ionizing radiation have an impact on the cell cycle and may induce cell cycle arrest or apoptosis. Mutations of the p53 gene are observed at a high frequency in human tumours and are recognized in about half of all human cancers. Sensitivity to radiation, heat and anticancer agents has been observed in p53(+/+) cells, but not in mutated or p53-deficient cells. Moreover, enhancement of radiosensitivity by HS has been observed in wild-type p53 cells but not in p53-deficient cells. The molecular mechanism of the differential cell response to HS or ionizing radiation is not yet understood. MATERIALS AND METHODS: Differences in cellular response to radiation (200 kV X-ray, 1, 2, 5 Gy) and HS (39 degrees C, 41 degrees C and 43 degrees C for 30 min) on cell cycle progression of cultures of human p53 mutant cells were investigated by flow cytometry. In addition, the effects of stressors used on the expression of several heat shock genes (HSP27, HSP60, HSP70, HSC70, HSP75, HSP78, HSP90) were studied by reverse transcriptase-polymerase chain reaction. RESULTS AND CONCLUSIONS: Yet, with respect to HSP gene expression, different stressors produced similar effects. Combination of HS and radiation treatment significantly induced the transcription of the HSP70 gene above the level induced by each stressor alone. Cell cycle analysis, however, revealed striking differences in prolonged dynamics of cell division in response to each stressor. Thus, p53 status could be a useful indicator in predictive assays for hyperthermia cancer treatment in combination with radiation and/or chemotherapy.

[The combained effects of the long-term gamma-irradiation and heavy metall ions on the haematopoietic system of rats].

The combiened effects of different dose rates (0.625 microGy/s - 1.1 mGy/s) of gamma-irradiation and of cuprum and of cadmium ions on the haematopoietic system of rats were studied. It was found that only low dose rates (0.625-10 microGy/s, summary doses 0.5-2.0 Gy) of gamma-irradiation yields in the increasing proliferative activity of bone marrow. The number of myelocariocytos in S-phase was increased at 1.5-1.8 times. In case of the treatment with both cadmium chloride and radiation the changes in proliferative activity of bone marrow are completely due to the radiation factor. Combination of cuprum acetate and ionizing radiation induce opposite effects providing formal normalization of the haematopoietic characteristic of bone marrow up to 3, 6 and 12 months after the end of the radiation and the chemical exposure of the animal.

Identification of actin as quercetin-binding protein: an approach to identify target molecules for specific ligands.

The biological effect of flavonoids is commonly studied by assaying the activity of a protein of interest. Taking a reverse approach, we identified target proteins of the widely studied flavonol quercetin by exploiting the altered spectroscopic properties of target proteins and ligands on their molecular interaction. Nuclear extracts of human leukemia cells were fractionated by column chromatography and assayed for their ability to alter the fluorescence emission spectra, and finally the proteins present in fractions of interest were identified by mass spectrometry. Among the identified proteins, actin was shown to be a quercetin-binding nuclear protein.

Spermatogenesis in testis primary cell cultures of the tilapia (Oreochromis niloticus).

Spermatogenesis in vertebrates is controlled by endocrine and paracrine factors and involves the communication between somatic and germ line cells. To elucidate some of the relevant factors in the complicated molecular control processes, we established an in vitro test system using primary cultures of tilapia (Oreochromis niloticus) testis cells. The cultures were enriched for germ line cells and Sertoli cells and largely depleted of spermatozoa. By staining the cells with propidium iodide and carboxyfluorescein succinimidyl ester (CFSE), different cell populations could be identified cytologically and, in addition, quantified by flow cytometry. Cells that had gone through one or more divisions could be identified unequivocally based on their CFSE staining intensity. In parallel cultures maintained for up to 16 days in the presence of 11-ketotestosterone (KT), insulin-like growth factor I (IGF), and/or human chorionic gonadotropin (hCG) the initiation of meiotic and mitotic divisions was monitored. Although KT was important for the initiation of meiosis, spermatogonial mitotic divisions between 10 days and 16 days of culture were promoted by IGF and/or hCG in the presence of KT. These results illustrate the potential of the established in vitro test system for the analysis of the molecular control mechanisms of spermatogenesis.

[Fluorescence of meso-tetrakis(4-(carboxy)phenyl)porphine covalently bound to oligonucleotides d(CG)5 and d(TA)5]. / Fluorestsentsiia mezo-tetrakis(4-(karboksi)fenyl)porfina, kovalentno sviazanogo s oligonukleotidami d(CG)5 i d(TA)5.

The amino-reactive derivative of tetraphenylporphine meso-tetrakis[4-(carboxy)phenyl]porphine (TCPP) was synthesized, which is characterized by a high molar absorption coefficient (epsilon 416 = 36,500 M-1.cm-1). TCPP was covalently attached to oligonucleotides d(CG)5 [d(CG)5-TCPP] and d(TA)5 [d(TA)5-TCPP]. The spectral characteristics of these complexes were studied in 0.01 M phosphate buffer, pH 7 at 23 degrees C. UV-visible absorption spectra of these complexes have a clearly pronounced Soret band at (414 +/- 1) nm for d(CG)5-TCPP and at (412 +/- 1) nm for d(TA)5-TCPP. The fluorescence spectra of these complexes have maxima at (648 +/- 2) nm for d(CG)5-TCPP and at (658 +/- 2) nm for d(TA)5-TCPP. In this study we also determined fluorescence quantum yields q and fluorescence lifetimes tau [q = 0.099 +/- 0.011, tau = (9.0 +/- 0.3) ns for d(CG)5-TCPP and q = 0.080 +/- 0.011, tau = (8.7 +/- 0.3) ns for d(TA)5-TCPP]. A temperature rise from 5 to 50 degrees C produced only slight (within 23%) emission changes in both samples studied. Taking into account: a) high fluorescence yields (q), b) weak dependence of q on temperature, c) weak q dependence of q on the oligonucleotide type, we conclude that TCPP may be used as a sensitive fluorescence label in DNA studies.

In vitro suppression of thymocyte apoptosis by metal-rich complex environmental mixtures: potential role of zinc and cadmium excess.

Excessive amounts of heavy metals (e.g. Zn, Cu, Mn, Cr) are accumulated in river bottom sediments (RBS), being available to humans and animals along food chains. Increased exposure of mammals to certain metals (Cr, Cu) induces immunosuppresion, due to DNA damage and decreased survival of lymphoid cells. By contrast, excess of Zn and Cd causes inhibition of apoptosis thus suggesting increased survival of genetically mutated cells and higher cancer risks in exposed populations. Rat thymic lymphocytes represent a well-established model for apoptosis testing. The primary goal of our study was to assess the degree of apoptosis modulation with a number of RBS extracts differing in their metal contents. A series of freshly deposited RBS was collected at nine sampling stations along the Elbe River. All sediments were rich in Fe, Mn and Zn. The contents of Cu, Cr, Ni, Cd, Hg, Pb and As were much lower and interrelated. The short-term cytotoxicity of aqueous sediment extracts was assessed, using the following criteria: total cell counts; incidence of apoptosis and necrosis (morphological detection by fluorescent microscopy); and nuclear chromatin decay (by DNA flow cytometry). RBS extracts produced both apoptosis and necrosis of thymocytes. High contents of zinc and other heavy metals in the samples correlated with decreased thymocyte apoptosis (r= -0.543 to -0.608, P <0.01). The rates of thymocyte damage showed a distinct dependence on the time and region of sampling. Apoptosis modulation was also tested with pure salts of Mn(II), Zn(II), Cu(II), Cr(III) and Cd(II), at the test concentrations of 1, 10 and 100 microM. Cu(II) and Cr(III) proved to induce marked dose-related apoptosis, whereas Zn(II) ions caused significant suppression of apoptosis. These effects were similar to those trends observed with metal-rich sediments. In the present study. DNA flow cytometry proved to be a less sensitive index of cell death than morphological assay of apoptosis and/or necrosis. In summary, inhibition of lymphocyte apoptosis by RBS extracts and pure metals is associated with excess of zinc and, probably, cadmium. The proposed model of lymphoid cell apoptosis is a promising tool for screening cytotoxic effects of complex environmental samples.

[Cerebrospinal fluid sorption in patients with heroin addiction]. / Likvorosorbtsiia u bol'nykh geroinovoi narkomaniei.

The sorption of cerebrospinal fluid (CSF) was attempted as a special detoxifying procedure in a group of sixty heroine addicts. CSF contents of cells, total protein, nucleic acids and interleukin-1 (IL-1), as well as acridine orange (AO) binding to CSF cells were determined before and after the procedure. The treatment provided immediate clinical improvement for 70% of the patients. Clinical effect was accompanied by decreased of CSF cells, diminished nucleic acids and protein amounts, along with increased DNA-AO binding and accumulation of IL-l. These data are interpreted in terms of intensive apoptosis of CSF cells and increased acute phase of aseptic inflammation-like events induced by the procedure of liquor sorption.

[The effect of soil extracts with different heavy metal levels on the viability of isolated blood system cells]. / Vliianie ékstraktov pochv s razlichnym soderzhaniem tiazhelykh metallov na zhiznesposobnost' izolirovannykh kletok sistemy krovi.

Short-term incubation of rat thymocytes, bone marrow cells, and macrophages with aqueous extracts of soil demonstrated positive correlations between damage to the cells and increased levels of copper, chromium, and manganese in the soil, while increased levels of zinc and lanthanum were associated with less pronounced changes in the cells.

[Effect of chronic irradiation on the cell cycle of rat myelokaryocytes]. / Vliianie khronicheskogo oblucheniia na kletochnyi tsikl mielokariotsitov krys.

Rats were exposed to gamma-radiation within dose range from 0.12 to 4.0 Gy at dose rates from 0.625 microGy/s to 1.1 mGy/s. The changes in myelokaryocyte distribution within the cell cycle phases during the period of 1 to 18 months after irradiation were studied by the method of flow cytometry. A dose-dependent increase of cell percentage in S-phase after irradiation at low dose rates (from 0.625 to 10.0 microGy/s) was found. The effect was kept till the end of irradiated rats life.

[The possibility of using the population characteristics of myelokaryocytes for the early diagnosis of radiation lesions]. / O vozmozhnosti ispol'zovaniia populiatsionnykh kharakteristik mielokariotsitov dlia rannei diagnostiki luchevykh porazhenii.

The method of flow cytofluorimetry was used to study some population characteristics of bone marrow cells of control and irradiated rats. The simultaneous recording of cellularity and distribution of myelokaryocytes among the cell cycle phases was shown to give reliable results for determining the extent to which the organism was exposed at early times following irradiation.

[The mechanism of the formation of a circadian rhythm of bone marrow proliferation in rats]. / O mekhanizme formirovaniia sutochnogo ritma proliferatsii kostnogo mozga u krys.

Flow cytofluorimetry and statmokinetic method were used to study the circadian rhythm of bone marrow proliferation in Pliss' lymphosarcoma-bearing and intact rats. These data were compared to those obtained in the study of the mitotic activity of the bone marrow in cancer patients. It was found that, already at early stage, tumor affected the circadian rhythm of bone marrow proliferation, reducing the amplitude of oscillations. A model simulating formation of the circadian rhythm of the bone marrow was suggested basing on the possibility to arrest cells at the end of G1 phase. The rate of transition of G1 cells to S phase was determined not only by endogenous "set-points" of the rhythm which formed the basic wave of proliferation but also by conditions of animal upkeep.