Clearance of radiation-induced apoptotic lymphocytes: ex vivo studies and an in vitro co-culture model

Lymphocytes are very sensitive to radiation. Our aim was to test the possibility of detecting apoptosis in lymphocytes as a potential short-term biomarker of ionizing radiation exposure. Our in vitro data confirmed the dose-time-effect relationships involved in radiation-induced apoptosis. The detection of in vivo induction of apoptosis in circulating lymphocytes after exposure of animals to radiation appears to depend critically on the technique used to measure apoptosis. Among the different techniques we investigated, mitochondrial modification was the most appropriate; they allowed establishment of dose-time-effect relationships when animals were observed for 72 h. A model of in vitro phagocytosis of apoptotic lymphocytes by macrophages was developed to mimic clearance of apoptotic cells occurring in vivo. Together, our data show that mitochondrial labeling may make it possible to detect ex vivo radiation-induced apoptosis of lymphocytes before macrophage ingestion occurs. We propose the measurement of apoptosis in lymphocytes as a potential short-term biomarker of ionizing radiation exposure.     

Effects of systemic glucocorticosteroids on peripheral neutrophil functions in asthmatic subjects: an ex vivo study

In 21 asthmatic subjects, several functions of isolated peripheral neutrophils (chemokinesis and chemotaxis toward 10% E. coli; superoxide anion generation after PMA; leukotriene B(4) (LTB(4)) release from whole blood and isolated neutrophtls, before and after different stimuli) were evaluated during an acute exacerbation of asthma, and after 14 - 54 days of treatment with systemic glucocorticosteroids (GCS). During acute exacerbation, superoxide anion generation was higher in asthmatics than in eleven normal subjects (39.2 +/- 14.1 vs. 25.2 +/- 7.3 nmol, p < 0.05); there was a significant correlation between FEV(1) (% of predicted) and neutrophil chemotaxis (r = -0.52, p = 0.04). After treatment, there was no significant change in all neutrophil functions, except for a decrease in neutrophil chemotaxis in subjects who showed an FEV(1) increase > 20% after GCS treatment (from 131 +/- 18 to 117 +/- 21 mum, p = 0.005). Chemokinesis sicantly decreased in all subjects, and the changes significantly correlated with an arbitrary score of the total administered dose of GCS (r = 0.57, p < 0.05). These data suggest that neutrophil activation plays a minor role in asthma, and that treatment with GCS is not able to modify most functions of peripheral neutrophils in asthmatic subjects; chemotaxis seems to be related only to the severity of the asthma and it could reflect the improvement of the disease.     

In vivo analysis of glial cell phenotypes during a viral demyelinating disease in mice

C57 BL/6N mice injected intracranially with the A59 strain of mouse hepatitis virus exhibit extensive viral replication in glial cells of the spinal cord and develop demyelinating lesions followed by virus clearing and remyelination. To study how different glial cell types are affected by the disease process, we combine three-color immunofluorescence labeling with tritiated thymidine autoradiography on 1-micron frozen sections of spinal cord. We use three different glial cell specific antibodies (a) to 2',3' cyclic-nucleotide 3' phosphohydrolase (CNP) expressed by oligodendrocytes, (b) to glial fibrillary acidic protein (GFAP) expressed by astrocytes, and (c) the O4 antibody which binds to O-2A progenitor cells in the rat. These progenitor cells, which give rise to oligodendrocytes and type 2 astrocytes and react with the O4 antibody in the adult central nervous system, were present but rare in the spinal cord of uninfected mice. In contrast, cells with the O-2A progenitor phenotype (O4 + only) were increased in number at one week post viral inoculation (1 WPI) and were the only immunostained cells labeled at that time by a 2-h in vivo pulse of tritiated thymidine. Both GFAP+ only and GFAP+, O4+ astrocytes were also increased in the spinal cord at 1 WPI. Between two and four WPI, the infected spinal cord was characterized by the loss of (CNP+, O4+) oligodendrocytes within demyelinating lesions and the presence of O-2A progenitor cells and O4+, GFAP+ astrocytes, both of which could be labeled with thymidine. As remyelination proceeded, CNP immunostaining returned to near normal and tritiated thymidine injected previously during the demyelinating phase now appeared in CNP+ oligodendrocytes. Thus O4 positive O-2A progenitor cells proliferate early in the course of the demyelinating disease, while CNP positive oligodendrocytes do not. The timing of events suggests that the O-2A progenitors may give rise to new oligodendrocytes and to type 2 astrocytes, both of which are likely to be instrumental in the remyelination process.     

Astroglial injury in an ex vivo model: contributions to its analysis in enriched cell cultures

In vitro cell culture models have been proposed to analyze some of the complex structural and functional characteristics involved in astroglial changes after neural injury in vivo. This report contributes to analyze the proposed hypothesis that an experimentally induced discontinuity of a confluent cellular culture could represent a useful model for the analysis of the processes involved in a neural lesion. For this purpose, it was decided to characterize astroglial proliferation and dye coupling state after a “scratch wound” applied to confluent, astrocyte-enriched cell cultures, obtained from several rat brain regions. Proliferation was assessed in terms of bromodeoxyuridine nuclear incorporation as a function of lesion width, serum deprivation, time after confluence, brain region origin, postlesional culture medium changes and agitation, and after application of a gap-junction uncoupling agent. The proliferative reaction after injury was neither cell type-specific nor brain region specific, nor was significantly affected by neither of the above-mentioned variables. Furthermore, injury failed to significantly affect the astroglial dye coupling state. Results suggest that the proliferative response observed under present conditions would depend on the disruption of contact inhibition rather than on astroglial mitogenic signals released from the wound and operating by either extracellular or cell coupling mechanisms. Present results question the validity of astrocyte-enriched cell cultures as an experimental model of neural tissue injury in vivo, as they do not appear to reproduce fundamental characteristics expressed in situ.     

MRI quantification in vivo of corticosteroid induced thymus involution in mice: correlation with ex vivo measurements

Thymus involution is a useful marker of transactivation-mediated side effects in preclinical therapeutic index testing of new anti-inflammatory glucocorticosteroids, and is usually measured post mortem. We have validated the use of MRI for non-invasive in vivo measurement of mouse thymus involution induced by dexamethasone (DEX). Tl-weighted spin echo 7 T images provided satisfactory contrast between thymus and surrounding connective tissue and fat. Increasing doses of DEX caused thymus involution, reflected in MRI volume (87+/-14, 33+/-10, 28+/-6, 16+/-7 microl in dosage groups of Cremophor vehicle, 1, 10 and 30 mg/kg subcutaneous respectively, n=6/group, mean+/-standard deviation) and post mortem wet weight (64+/-12, 33+/-6, 25+/-9, 23+/-8 mg). Correlation between MRI volumes and wet weights was very good (r=0.842). Measuring pre-dose MRI volumes and then assessing DEX effects as post-dose change from baseline produced no statistical advantage relative to considering post-dose MRI thymus volume alone, probably due to variability in pre-dose baseline values compounding post-dose variability. Smaller group sizes were sufficient to achieve a given statistical power using MRI post-dose volume than using wet weight, suggesting a role for MRI in differentiating the effects of compounds which produce similar effects, or in contexts where the use of large groups of animals is impractical or ethically unacceptable.     

Maternal hyperthermia disrupts developmental competence of follicle-enclosed oocytes: in vivo and ex vivo studies in mice

Mammalian oocytes are susceptible to thermal stress at various stages of follicular development. We examined whether the ovarian pool of oocytes is susceptible to maternal hyperthermia and if so, whether hyperthermia at the germinal vesicle (GV) stage further affects the developmental competence of preimplantation embryos and offspring quality. Synchronized female mice were exposed to thermal stress (40 degrees C, 65% RH) for 1.5-2h or maintained under normothermal conditions (25 degrees C, 45% RH). Thereafter, mice were paired with stud males. In the first experiment, mated mice were sacrificed 20h post hCG administration, and in vivo-derived zygotes were recovered and cultured in vitro. Maternal hyperthermia decreased the percentage of putative zygotes of apparent normal morphology in the heat-stressed group (81+/-1.3%) as compared to the control group (86+/-1.2%). Developmental competence was also compromised as expressed by the disruption in cleavage timing pattern, resulting in a reduced developmental rate to the blastocyst stage (57+/-2.6% versus 84+/-1.9%). In the second experiment, both groups were left with stud males until litter delivery. Litter size in the first delivery cycle was lower for the heat-stressed group (7.7+/-1.1 pups), followed by a slight increase throughout consecutive cycles as compared to the control group (11.3+/-1.0 pups). Behavioral examinations of 8-week-old pups revealed similar locomotor activity and learning potential between the groups. In summary, the findings indicate that a subpopulation of the ovarian pool of follicles is highly sensitive to thermal stress and that maternal hyperthermia disrupts developmental competence of GV-stage oocytes. Pups that developed from oocytes that survived thermal stress exhibited a developmental potential similar to that of the of control pups.     

Structure-function analysis from the outside in: long-range tertiary contacts in RNA exhibit distinct catalytic roles

The conserved catalytic core of the Tetrahymena group I ribozyme is encircled by peripheral elements. We have conducted a detailed structure-function study of the five long-range tertiary contacts that fasten these distal elements together. Mutational ablation of each of the tertiary contacts destabilizes the folded ribozyme, indicating a role of the peripheral elements in overall stability. Once folded, three of the five tertiary contact mutants exhibit defects in overall catalysis that range from 20- to 100-fold. These and the subsequent results indicate that the structural ring of peripheral elements does not act as a unitary element; rather, individual connections have distinct roles as further revealed by kinetic and thermodynamic dissection of the individual reaction steps. Ablation of P14 or the metal ion core/metal ion core receptor (MC/MCR) destabilizes docking of the substrate-containing P1 helix into tertiary interactions with the ribozyme's conserved core. In contrast, ablation of the L9/P5 contact weakens binding of the guanosine nucleophile by slowing its association, without affecting P1 docking. The P13 and tetraloop/tetraloop receptor (TL/TLR) mutations had little functional effect and small, local structural changes, as revealed by hydroxyl radical footprinting, whereas the P14, MC/MCR, and L9/P5 mutants show structural changes distal from the mutation site. These changes extended into regions of the catalytic core involved in docking or guanosine binding. Thus, distinct allosteric pathways couple the long-range tertiary contacts to functional sites within the conserved core. This modular functional specialization may represent a fundamental strategy in RNA structure-function interrelationships.     

Targeting myeloid leukemia with a DT(390)-mIL-3 fusion immunotoxin: ex vivo and in vivo studies in mice.

The IL-3 receptor was expressed on a high frequency of myeloid leukemia cells and also on hematopoietic and vascular cells. We previously showed that a recombinant IL-3 fusion immunotoxin (DT(390)IL-3) expressed by splicing the murine IL-3 gene to a truncated diphtheria toxin (DT(390)) gene selectively killed IL-3R(+) expressing cells and was not uniformly toxic to uncommitted BM progenitor cells (Chan,C.-H., Blazar,B.R., Greenfield,L., Kreitman,R.J. and Vallera,D.A., 1996, Blood, 88, 1445-1456). Thus, we explored the feasibility of using DT(390)IL-3 as an anti-leukemia agent. DT(390)IL-3 was toxic when administered to mice at doses as low as 0.1 microg/day. The dose limiting toxicity appeared to be related to platelet and bleeding effects of the fusion toxin. Because of these effects, DT(390)IL-3 was studied ex vivo as a means of purging contaminating leukemia cells from BM grafts in a murine autologous BM transplantation. In this setting, as few as 1000 IL-3R-expressing, bcr/abl transformed myeloid 32Dp210 leukemia cells were lethal. An optimal purging interval of 10 nM/l for 8 h eliminated leukemia cells from 32Dp210/BM mixtures given to lethally irradiated (8 Gy) C3H/HeJ syngeneic mice. Mice given treated grafts containing BM and a lethal dose of 32Dp210 cells survived over 100 days while mice given untreated grafts did not survive (P < 0.00001). DT(390)IL-3 may prove highly useful for ex vivo purging of lethal malignant leukemia cells from autologous BM grafts.     

Abiotic elicitors mediated elicitation of innate immunity in tomato: an ex vivo comparison

Improvement of the host resistance by using hazard free chemical elicitors is emerging as an alternative approach in the field of plant disease management. In our present work, we have screened the efficacy and possible mechanism of abiogenic elicitors like Dipotassium hydrogen orthophosphate (K₂HPO₄), Oxalic acid (OA), Isonicotinic acid (INA), Salicylic acid (SA), Acetylsalicylate (AS), Arachidonic acid (AA) and Calcium chloride (CaCl₂) to stimulate innate immune responses in Lycopersicum esculentum Mill. Excised tomato leaves, treated with elicitors at three different concentrations, were found to stimulate defense and antioxidative enzymes, total phenol and flavonoid content after 24 h of incubation. CaCl₂ (0.5 %) followed by INA (2.5 mM) were found most effective in activation of all such defense molecules in tomato leaves. Furthermore, nitric oxide (NO), a key gaseous mediator in plant defense signaling, was also measured after subsequent elicitor application. Higher doses of elicitors showed an elevated level of reactive oxygen species (ROS) generation, enhanced lipid peroxidation rate and proline content, which indicates the extent of abiotic stress generation on the leaves. However, ROS production, lipid peroxidation rate and proline concentration remain significantly reduced as a result of CaCl₂ (0.5 %) and INA (2.5 mM) application. A sharp increase of total chlorophyll content was also recorded due to treatment of CaCl₂ (0.5 %). These results demonstrate the effects of different abiogenic elicitors to regulate the production of defense molecules. Results also suggest that among all such chemicals, CaCl₂ (0.5 %) and INA (2.5 mM) can be used as a potential elicitor in organic farming of tomato.     

Intraocular pressure in genetically distinct mice: an update and strain survey

Background  

Little is known about genetic factors affecting intraocular pressure (IOP) in mice and other mammals. The purpose of this study was to determine the IOPs of genetically distinct mouse strains, assess the effects of factors such as age, sex and time of day on IOP in specific strain backgrounds, and to assess the effects of specific candidate gene mutations on IOP.     

Protein kinase C modulation in apoptotic rat thymocytes: an ultrastructural analysis

Numerous events in the cell, such as gene expression, cell growth and metabolism are regulated by signal transduction pathways involving protein kinase C (PKC). Recent data indicate that a PKC-dependent mechanism also underlies the apoptotic death of cells induced by glucocorticoid hormones. In this report we have analysed the changes of PKC during dexamethasone-induced apoptosis in thymocytes by means of immunocytochemical and immunochemical analysis. The data obtained show an increase and intracellular movement of protein kinase C, which is translocated to the nucleus and linked to the nuclear matrix during the apoptotic process.     

Evaluation of copper toxicity in isolated human peripheral blood mononuclear cells and it's attenuation by zinc: ex vivo

Copper and zinc act as a cofactor of over 300 mammalian proteins. Both have same electronic configuration therefore they are antagonist at higher individual concentration. The present study was designed with the aim to investigate the mechanisms pertaining to toxic effects of copper on human peripheral blood mononuclear cells (PBMCs) and to evaluate the cytoprotective effect of zinc on copper-induced cytotoxicity. The copper uptake into PBMCs was progressively increased with increasing concentration of metal in the growth medium. However, no significant effect on copper uptake was observed in the presence of zinc. Cell proliferation rate was decreased with increasing copper concentration. Interestingly, the proliferation rate of zinc treated PBMCs remained nearly the same as that of control cells. LD₅₀ of copper (115 μM) was increased six times (710 μM) in presence of zinc for PBMCs. At higher concentrations of copper (> 100 μM) decrease level of GSH was noticed. Increased levels of metallothionein in PBMCs were observed in response to zinc. DNA fragmentation studies also showed that copper produced DNA fragmentation at LD₅₀ (115 μM). Subsequently, zinc showed protection against DNA fragmentation caused by copper. Cell structure of PBMCs at LD₅₀ (115 μM copper) showed membrane bound cystic spaces and mitochondria having disrupted cristae and few myelin figures. In presence of zinc at LD₅₀ of copper (115 μM) cells showed improvement in mitochondrial structure and membrane bound cystic spaces. Taken together, the results of our study demonstrates that zinc play an important role in prevention of copper toxicity in peripheral blood mononuclear cells.     

Flow cytometric analysis of peripheral blood erythrocyte chimerism in alpha-thalassemic mice.

A rapid and reliable method for longitudinal studies on the degree of red cell chimerism following bone marrow transplantation of alpha-thalassemic recipient mice is presented. Blood obtained by tail clipping from transplanted mice was analyzed by measuring forward light scatter (FLS) distribution of red cells using a flow cytometer. Amplification and threshold of FLS were specifically adjusted. For flow cytometric analysis, the red cells needed to be suspended in hypotonic saline (103 mmol/l NaCl). Osmotic fragility testing showed that lysis of erythrocytes did not significantly influence the measurements. Flow cytometric measurement allowed for a rapid determination of the degree of red cell chimerism.     

Cytochemical analysis of organelle degradation in phagosomes and apoptotic cells of the mucoid epithelium of mice.

The activity of mitochondrial cytochrome oxidase and peroxisomal catalase in the phagolysosomes and apoptotic bodies of mucoid epithelial cells was analysed. Tissue from 2-6 day old mice was used. The activity of acid phosphatase in lysosomes was also estimated. Cytochrome oxidase was demonstrated in well-preserved mitochondria inside phagosomes. Mitochondria in cells exhibiting apoptotic death also show activity of cytochrome oxidase. The enzyme activity in swollen mitochondria ceases before the membranes of the cristae disappear completely. Apoptotic bodies are phagocytosed by sister mucoid cells and, later on, they are digested inside the cell. Phagosomes which contain already degraded mitochondria show still active catalase in sequestered peroxisomes. The acid phosphatase involved in degradation of phagocytosed material originates from endocytosed lysosomes and primary and secondary lysosomes which fuse with the membranes of phagosomes.