Ewa Jasek

Effect of differentiating agents (all-trans retinoic acid and phorbol 12-myristate 13-acetate) on drug sensitivity of HL60 and NB4 cells in vitro.

In vitro studies have shown that human myeloid leukemia cell lines: HL60 and NB4 can be stimulated to differentiation by various agents, for example, all-trans retinoic acid (ATRA) and phorbol 12-myristate 13-acetate (PMA). The purpose of this study was to investigate whether differentiation of HL60 and NB4 leukemia cell lines induced by ATRA and PMA alters their drug sensitivity. The differentiation along the neutrophil lineage (upon stimulation with ATRA) and along the monocyte/macrophage lineage (upon stimulation with PMA) was proved by decreased proliferative potential of cells, changes in their morphology, increased ability for NBT reduction and increased expression of CD11b and CD14 cell surface markers. The effect of drugs: cytosine arabinoside, daunorubicin, mitoxantrone and etoposide was examined by Alamar Blue test (proliferation and survival rates), as well as by evaluation of cell smears stained with Hoechst 33342 (apoptotic index). Differentiation resulted in the change of drug sensitivity in both cell lines: the differentiation along the neutrophil pathway (after stimulation with ATRA) increased sensitivity to cytosine arabinoside and mitoxantrone but decreased sensitivity to etoposide; the differentiation along the monocyte/macrophage pathway (induced by PMA) resulted in the decreased sensitivity of both cell lines to all drugs tested. In conclusion, we have shown that ATRA- and PMA-mediated differentiation of HL60 and NB4 cell lines results in the changes of their drug sensitivity. Our data may provide a contribution to a strategy aimed at a rational combination of differentiating agents and conventional anticancer drugs.

Circadian rhythmicity of synapses in mouse somatosensory cortex.

The circadian rhythmicity displayed by motor behavior of mice: activity at night and rest during the day; and the associated changes in the sensory input are reflected by cyclic synaptic plasticity in the whisker representations located in the somatosensory (barrel) cortex. It was not clear whether diurnal rhythmic changes in synapse density previously observed in the barrel cortex resulted from changes in the activity of the animals, from daily light/dark (LD) rhythm or are driven by an endogenous clock. These changes were investigated in the barrel cortex of C57BL/6 mouse strain kept under LD 12 : 12 h conditions and in constant darkness (DD). Stereological analysis of serial electron microscopic sections was used to assess numerical density of synapses. In mice kept under LD conditions, the total density of synapses and the density of excitatory synapses located on dendritic spines was higher during the light period (rest phase). In contrast, the density of inhibitory synapses located on dendritic spines increased during the dark period (activity phase). Under DD conditions, the upregulation of the inhibitory synapses during the activity phase was retained, but the cyclic changes in the density of excitatory synapses were not observed. The results show that the circadian plasticity concerns only synapses located on spines (and not those on dendritic shafts), and that excitatory and inhibitory synapses are differently regulated during the 24 h cycle: the excitatory synapses are influenced by light, whilst the inhibitory synapses are driven by the endogenous circadian clock.

Fear learning increases the number of polyribosomes associated with excitatory and inhibitory synapses in the barrel cortex.

Associative fear learning, resulting from whisker stimulation paired with application of a mild electric shock to the tail in a classical conditioning paradigm, changes the motor behavior of mice and modifies the cortical functional representation of sensory receptors involved in the conditioning. It also induces the formation of new inhibitory synapses on double-synapse spines of the cognate barrel hollows. We studied density and distribution of polyribosomes, the putative structural markers of enhanced synaptic activation, following conditioning. By analyzing serial sections of the barrel cortex by electron microscopy and stereology, we found that the density of polyribosomes was significantly increased in dendrites of the barrel activated during conditioning. The results revealed fear learning-induced increase in the density of polyribosomes associated with both excitatory and inhibitory synapses located on dendritic spines (in both single- and double-synapse spines) and only with the inhibitory synapses located on dendritic shafts. This effect was accompanied by a significant increase in the postsynaptic density area of the excitatory synapses on single-synapse spines and of the inhibitory synapses on double-synapse spines containing polyribosomes. The present results show that associative fear learning not only induces inhibitory synaptogenesis, as demonstrated in the previous studies, but also stimulates local protein synthesis and produces modifications of the synapses that indicate their potentiation.

Microvessel Density and Area in Pituitary Microadenomas

Microvessel density (MVD) and area (MVA) were assessed in 53 clinically silent pituitary microadenomas and in surrounding normal pituitary tissue using CD34 immunostaining and digital image analysis system. Twenty-one microadenomas (40%) were avascular; in the others, both MVD and MVA were significantly lower than in the normal pituitary tissue. No significant differences in MVD and MVA were found between hormonally immunonegative and immunopositive tumors or between different hormonal subtypes of immunopositive microadenomas. In microadenomas and in normal pituitary tissue, MVD and MVA were not significantly influenced by age or sex. These results suggest that pituitary adenomas at early developmental stage are either avascular or significantly less vascular than normal pituitary tissue and that differences in vascularity of pituitary adenomas associated with the variables of sex, age, clinical presentation and immunohistochemical phenotype reported in the literature can evolve with tumor growth and possibly acquire statistical significance in large macroadenomas.

Vascular Effects of Advanced Glycation End-Products: Content of Immunohistochemically Detected AGEs in Radial Artery Samples as a Predictor for Arterial Calcification and Cardiovascular Risk in Asymptomatic Patients with Chronic Kidney Disease

Objectives. Our aim was to determine whether vascular deposition of advanced glycation end-products (AGEs) is associated with arterial calcification and cardiovascular mortality in chronic kidney disease (CKD) patients and to assess the relationships between vascular content of AGEs and selected clinical and biochemical parameters. Materials and Methods. The study comprised 54 CKD patients (33 hemodialyzed, 21 predialyzed). Examined parameters included BMI, incidence of diabetes, plasma fasting glucose, AGEs, soluble receptor for AGEs and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging, serum C-reactive protein (hsCRP), plasminogen activator inhibitor-1 (PAI-1), and fetuin-A. Fragments of radial artery obtained during creation of hemodialysis access were stained for calcifications using alizarin red. AGEs deposits were identified immunohistochemically and their relative content was quantified. Results. Vascular content of AGEs was positively correlated with BMI, hsCRP, fetuin-A, PAI-1, and DPPH scavenging in simple regression; only fetuin-A was an independent predictor in multiple regression. There was a significant positive trend in the intensity of AGEs immunostaining among patients with grades 1, 2, and 3 calcifications. AGEs immunostaining intensity predicted 3-year cardiovascular mortality irrespective of patients age. Conclusions. The present study demonstrates an involvement of AGEs in the development of medial arterial calcification and the impact of arterial AGE deposition on cardiovascular mortality in CKD patients.

Distribution of selected elements in calcific human aortic valves studied by microscopy combined with SR-μXRF: influence of lipids on progression of calcification.

Calcified heart valves display a significant imbalance in tissue content of trace and essential elements. The valvular calcification is an age-related process and there are data suggesting involvement of lipids. We studied elemental composition and lipid distribution in three distinct regions of calcified human aortic valves, representing successive stages of the calcific degeneration: normal, thickened (early lesion) and calcified (late lesion), using SR-μXRF (Synchrotron Radiation Micro X-Ray Fluorescence) for elemental composition and Oil Red O (ORO) staining for demonstration of lipids. Two-dimensional SR-μXRF maps and precise point spectra were compared with histological stainings on consecutive valve sections to prove topographical localization and colocalization of the examined elements and lipids. In calcified valve areas, accumulation of calcium and phosphorus was accompanied by enhanced concentrations of strontium and zinc. Calcifications preferentially developed in lipid-rich areas of the valves. Calcium concentration ratio between lipid-rich and lipid-free areas was not age-dependent in early lesions, but showed a significant increase with age in late lesions, indicating age-dependent intensification of lipid involvement in calcification process. The results suggest that mechanisms of calcification change with progression of valve degeneration and with age.

Effect of Associative Learning on Memory Spine Formation in Mouse Barrel Cortex

Associative fear learning, in which stimulation of whiskers is paired with mild electric shock to the tail, modifies the barrel cortex, the functional representation of sensory receptors involved in the conditioning, by inducing formation of new inhibitory synapses on single-synapse spines of the cognate barrel hollows and thus producing double-synapse spines. In the barrel cortex of conditioned, pseudoconditioned, and untreated mice, we analyzed the number and morphological features of dendritic spines at various maturation and stability levels: sER-free spines, spines containing smooth endoplasmic reticulum (sER), and spines containing spine apparatus. Using stereological analysis of serial sections examined by transmission electron microscopy, we found that the density of double-synapse spines containing spine apparatus was significantly increased in the conditioned mice. Learning also induced enhancement of the postsynaptic density area of inhibitory synapses as well as increase in the number of polyribosomes in such spines. In single-synapse spines, the effects of conditioning were less pronounced and included increase in the number of polyribosomes in sER-free spines. The results suggest that fear learning differentially affects single- and double-synapse spines in the barrel cortex: it promotes maturation and stabilization of double-synapse spines, which might possibly contribute to permanent memory formation, and upregulates protein synthesis in single-synapse spines.

Effect of cultured autologous oral keratinocyte suspension in fibrin glue on oral wound healing in rabbits

The effect of cultured autologous oral keratinocyte suspension in fibrin glue on the healing of surgically produced oral mucosal wounds was assessed in the rabbit model. Using the light microscope and a digital image analysis system, the epithelization parameters (marginal epithelization and percentage of wound re-epithelization) were measured in haematoxylin-eosin stained sections of the wound area and compared with those of wounds treated with fibrin glue alone and untreated ones. The epithelization was significantly higher in keratinocytes plus fibrin glue-treated wounds on postoperative days 3 and 7. No significant differences were observed on postoperative day 1, when the healing process had just begun, and on postoperative day 14, when re-epithelization was completed or nearly completed in all groups. The inflammatory infiltration of the wounded mucosa was weakest in keratinocyte-treated wounds and strongest in untreated wounds. In conclusion, suspension of cultured autologous oral keratinocytes in fibrin glue significantly accelerates oral wound healing in the rabbit model and could be beneficial in the treatment of oral wounds in patients.

Expression of basal cell marker revealed by RAM11 antibody during epithelial regeneration in rabbits.

RAM11 is a mouse monoclonal anti-rabbit macrophage antibody recognizing connective tissue and vascular macrophages. Our previous report showed that RAM11 reacted with basal cells of stratified squamous epithelia of rabbit skin, oral mucosa and esophagus. The aim of the present study was to follow the appearance of RAM11 immunoreactivity in basal cells of regenerating oral epithelium in rabbits. No RAM11 immunostaining was observed in the regenerating epithelium examined on days 1 and 3 of wound healing. A weak immunofluorescence first appeared on day 7 in single basal cells and 32% of RAM11- positive basal cells were observed on day 14. These findings indicate that expression of the antigen recognized by RAM11 antibody is a transient event in the differentiation of oral keratinocytes which not always occurs during epithelial repair, although it is a constant feature of epithelial turnover in mature epithelium. Therefore this antigen can be regarded as basal cell marker only in mature stratified squamous epithelia.