Distribution of estrogen receptor β mRNA-containing cells in ovariectomized and estrogen-treated female rat brain

Estrogen receptor (ER)-β is a member of the nuclear receptor superfamily and mediates various estrogenic actions. Changes in ER-α mRNA expression induced by estrogen have been well documented, whereas those with regard to ER-β have only been reported for a part of the hypothalamus. In the present study, we examined the effect of estrogen on ER-β mRNA expression in the female rat brain. Detection of ER-β mRNA using the in situ hybridization method with a digoxigenin-labeled RNA probe was performed in two groups of female rats: ovariectomized (OVX) and estrogen (E₂)-treated. A wide distribution of ER-β mRNA-containing cells was demonstrated in both groups. In the E₂-treated group compared with the OVX group, the number of ER-β mRNA-containing cells was significantly reduced in the external plexiform layer of the olfactory bulb, entorhinal cortex, intermediate part of the lateral septal nucleus, nucleus of the horizontal limb of the diagonal band, amygdala (lateral, medial and basolateral part), thalamus (anteroventral, laterodorsal and lateral posterior part), medial geniculate nucleus, suprachiasmatic nucleus and Purkinje cells in the cerebellum. These results reveal that ER-β mRNA-containing cells were decreased by estrogen in several brain regions in the female rat brain, suggesting that ER-β mRNA is downregulated by the physiological level of estrogen in a region-specific manner.     

17β-Estradiol-induced remodeling of GABAergic axo-somatic synapses on estrogen receptor expressing neurons in the anteroventral periventricular nucleus of adult female rats

Experimental data demonstrate that the nervous system is widely influenced by sex hormones and the brain is continuously shaped by the changing hormone milieu throughout the whole life. Earlier we demonstrated that on the effect of estradiol there is a cyclic synaptic remodeling, i.e. a transient decrease in the number of GABAergic axo-somatic synapses in the arcuate nucleus. By using preembedding estrogen receptor and postembedding GABA immunostaining, in the present paper we studied the specificity of this effect and we found that in the anteroventral periventricular nucleus (AvPv) of adult female rats 17β-estradiol treatment does not affect all synapses and neurons. In contrast to the arcuate nucleus, hormonal treatment induces a significant increase of inhibitory axo-somatic synapses in the AvPv and we found selectivity at the level of the postsynaptic neurons, as well. We analyzed the hormone-induced synaptic remodeling in estrogen receptor alpha and beta immunoreactive and non-labeled cells and the change in synapse number was observed only in neurons which express estrogen β receptor.     

Immunohistochemical study on caveolin-1α in regenerating process of tubular cells in gentamicin-induced acute tubular injury in rats

Caveolin-1, a principal component of caveolae, modulates growth signaling, endocytosis, and intracellular transport. We examined the expression of caveolin-1α and its relation to cell cycle and caveolin-interacting growth factor receptors in regenerating proximal tubules (PTs) after gentamicin-induced acute renal failure in rats. Caveolin-1α appeared in regenerating PTs as early as day 4 after last gentamicin, peaked at days 6 to 8, and showed cytoplasmic pattern after day 8. Immunoelectron microscopy revealed caveolin-1α-positive caveolae on the cell membrane and in cytoplasms in regenerating PTs at days 4 to 8 and caveolin-positivity confined to cytoplasms after day 10. The number of PT cells with proliferation markers peaked at day 6 and decreased afterwards as expression of cyclin-dependent kinase inhibitors increased. Platelet-derived growth factor receptor-β (PDGFR-β) and epidermal growth factor receptor (EGFR) were colocalized with caveolin-1α in proliferating PTs as early as day 4. Phosphorylated EGFR increased at day 8 and afterwards when caveolins dissociated from EGFR or decreased. In case of PDGFR-β, phosphorylation seemed to be associated with the increase and association of caveolins to the receptors. Our results suggest that transient expression of caveolin-1α in early regenerating PTs might contribute to the regenerating process of PTs through modulating growth factor receptors.     

Effects of peripheral κ opioid receptor activation on inflammatory mechanical hyperalgesia in male and female rats

Activation of peripheral κ opioid receptors (KOR) effectively relieves pain and hyperalgesia in preclinical and clinical models of pain. Although centrally located KOR activation results in sexually dimorphic effects, it is unclear whether peripheral KOR also produces sex dependent effects in persistent inflammatory pain conditions. In this study, we investigated whether local administration of a specific KOR agonist, U50, 488 relieve mechanical hyperalgesia induced by the injection of complete Freund's adjuvant (CFA) in the rat hindpaw, and whether there are sex differences. The effects of U50, 488 were assessed three days after the induction of CFA-induced inflammation, a time point at which mechanical hyperalgesia was most prominent. There were no sex differences in baseline and CFA-induced changes in mechanical thresholds between male and female rats. Local treatment of U50, 488 produced moderate, but significant, anti-hyperalgesia in both male and female rats. However, U50, 488 was significantly more effective in male rats at the highest dose of U50, 488. We confirmed that the highest dose of U50, 488 used in this study did not produce systemic effects, and that the drug effect is receptor specific. On the basis of these results, we suggest that local KOR agonists are effective in mitigating mechanical hyperalgesia under a persistent inflammatory pain condition and that sex differences in anti-hyperalgesic effects become more evident at high doses.     

FcϵRI on human Langerhans cells: a receptor in search of new functions

The recent demonstration that the high-affinity receptor for IgE, FcϵRI, is expressed on human Langerbans cells casts a new perspective on the function of this receptor. This observation also provides a new direction for further investigation into the pathophysiological relevance of Langerbans cells and other antigen-presenting cells in the development of atopic diseases.     

Immunohistochemical organization patterns of the follicular dendritic cells, myofibroblasts and macrophages in the human spleen��New considerations on the pathological diagnosis of splenectomy pieces

There is reliable information about how changes in spleen histology are influenced by the relationship among B and T lymphocytes, macrophages, dendritic cells and myofibroblasts. Moreover, if it can be applied in the day-by-day pathology laboratory. This work intends to elucidate morpho-functional aspects of relationships of these cells in the different spleen compartments, how they are influenced by pathological conditions and how basic immunohistochemical techniques could optimize the histopathological diagnosis. We analyzed the usefulness of the monoclonal antibodies CD45RO, CD20, CD21, CD35, CD68, caldesmon, the smooth muscle α-actin type 1 (SMA-1) in 91 specimens. CD21⁺ CD35⁺ follicular dendritic cells were organized into three patterns in agreement with the immune condition of the lymphoid follicle. Smooth muscle α-actin type 1⁺and caldesmon⁺myofibroblasts draw two double rings: marginal–perifollicular and germinal–marginal. The latter is closely related to T-cells. CD68⁺red pulp macrophages had clear and linear configuration. The interruption of this CD68⁺ linear pattern in splenic marginal zone lymphoma cases could be a criterion to differentiate it from reactive hyperplasia. CD45RO, CD20, CD21, CD68 and SMA-1 provide a basic and quality immunohistochemical battery for a better comprehension of the human spleen and could improve its histopathological diagnosis.     

Increase of bone marrow lymphocytes in systemic mastocytosis: reactive lymphocytosis or malignant lymphoma? Immunohistochemical and molecular findings on routinely processed bone marrow biopsy specimens

AIMS: To clarify the nature (reactive or neoplastic) of lesional, perifocally aggregated lymphocytes in bone marrow infiltrates of systemic mastocytosis (SM), the histopathology of which can resemble malignant lymphoma with focal bone marrow involvement, particularly low grade malignant B cell lymphoma of lymphoplasmacytic immunocytoma subtype, which frequently exhibits increased mast cell (MC) numbers. METHODS: Thirteen cases of SM and three of lymphoplasmacytic immunocytoma with predominant focal bone marrow infiltration were investigated. Immunostaining of formalin fixed, paraffin wax embedded bone marrow specimens was performed using antibodies against CD2, CD5, CD20, CD23, and CD25; kappa and lambda immunoglobulin light chains; and MC markers chymase, tryptase, and CD117 (KIT). Monoclonal rearrangements of IgH and TCRgamma were studied using seminested polymerase chain reaction (PCR). c-kit point mutation Asp816-Val was detected by PNA mediated PCR clamping and hybridisation probes. RESULTS: The lymphocytic clusters in SM contained nearly equal numbers of mature T and B cells, the latter with no coexpression of aberrant antigens, such as CD5 or CD23. Most MCs in SM cases constantly coexpressed tryptase, CD25, and CD117. No monoclonal rearrangements were seen for IgH or TCRgamma. In contrast, B cells from immunocytomas showed light chain restriction and monoclonal rearrangement for IgH, confirming their neoplastic nature. c-kit point mutation Asp816-Val was found in ten of 13 SM cases, but in none of the three immunocytomas. CONCLUSIONS: Focal accumulations of lymphocytes in the bone marrow of SM are reactive in nature and could be termed lymphocytosis. A diagnosis of SM-AHNMD/immunocytoma should not be made.     

Effect of neonatal exposure to 5-bromo-2′-deoxyuridine on life span,estrus function and tumor development in rats — an argument in favor of the mutation theory of aging?

Outbred LIO rats were exposed to subcutaneous injections (3.2 mg) of a synthetic analogue of thymidine, 5-bromo-2′-deoxyuridine (BrdUrd), on days 1 and 3, or days 1, 3, 7 and 21 of postnatal life. The mean life span decreased by 31% and 38% in male and by 14% and 27% in female rats that received 2 and 4 injections of BrdUrd, respectively, in comparison to untreated controls. The opening of the vagina was delayed, whereas age-related changes in the length of the estrous cycle and in the incidence of persistent estrus and/or anestrus were observed earlier in BrdUrd-injected female rats than in untreated ones. Inhibition of compensatory ovarian hypertrophy induced by hemiovariectomy at the age of 3 months was found in females exposed neonatally to BrdUrd as compared to untreated rats, while the uterus weight increase induced by the administration of human chorionic gonadotropin was similar in both control and BrdUrd-treated infantile rats. These data suggest that exposure to BrdUrd in early life impairs pituitary gonadotropic function in female rats. It was also shown that neonatal administration of BrdUrd to rats doubles the incidence of chromosome aberrations in peripheral blood lymphocytes in comparison to controls and is followed by a dose-related increase in tumor incidence. Our observations on the decrease in mean and maximum life span, acceleration of age-related changes in reproductive system function, increase in chromosome aberration and tumor incidence and decrease in tumor latency in rats exposed to BrdUrd in early life suggest that this model could be used as a model of accelerated aging and that some of the results can be interpreted as arguments in favor of the mutation theory of aging.     

Immunohistochemical studies on the proliferative marker Ki-67 and estrogen receptor alpha (ERα) in the uterus of neonatal and immature pigs following exposure to flutamide

The development of uterine glands is characterized by the proliferation of epithelial cells and by estrogen receptor alpha (ERα) expression in the nascent glandular epithelium. It is known that androgen receptors are present in the porcine uterus during prenatal development and the neonatal window, when adenogenesis occurs. Therefore, the objective of the study was to determine whether the effects of maternal or neonatal administration of the anti-androgen, flutamide, could entail changes in the presence of ERα and proliferation of uterine cells in neonatal and three-month-old pigs. Following prenatal flutamide exposure, morphological differences and the acceleration of uterus differentiation marked by ERα expression in epithelial crypts were observed in the neonatal piglets. In the three-month-old pig uterus, the proliferation of stromal cells was observed only after prenatal exposure to flutamide, whereas ERα staining was weaker. The neonatal administration of flutamide caused a significant decrease in the proliferation of the surface epithelium and diminished intensity of ERα staining in the stromal cells of the uterus of three-month-old pigs, which paralleled decreased estrogen levels in these animals. Overall, prenatal flutamide exposure promoted growth and development of the neonatal porcine uterus. Moreover, in three-month-old pigs, flutamide application during the neonatal period decreased surface epithelium proliferation and stromal ERα expression, which confirmed the importance of epithelial-stromal interactions in the adenogenesis.     

Effect of zoledronic acid and an antibody against bone sialoprotein II on MDA-MB-231GFP breast cancer cells in vitro and on osteolytic lesions induced in vivo by this cell line in nude rats

The aim of this study was to investigate the combined effect of zoledronic acid and an antibody against bone sialoprotein II (BSPII) on proliferation and osteolytic activity of MDA-MB-231^GFP breast cancer cells. For this purpose, the cells were exposed to zoledronic acid (10–20 μg/ml [25–50 μM]) and an anti-BSPII IgY (10–100 μg/ml) for up to 5 days alone or in combination. The combined treatment showed synergistic antiproliferative effects at the higher dose of zoledronic acid. Following inoculation of 1 × 10⁵ MDA-MB-231 ^GFP breast cancer cells into a branch of the femoral artery of nude rats, lytic lesions developed in the tibia, femur or fibula of the injected hind leg after approximately 30 days. The appearance and development of these lesions were monitored radiographically. Rats with lytic lesions were treated with zoledronic acid (60 μg/kg/week sc × 8; n = 10), zoledronic acid and an anti-BSPII IgY antibody (60 μg/kg/week sc × 8 + 10 mg/kg/week sc × 8; n = 10), or left untreated (n = 20). In addition, rats were treated for 4 weeks (n = 10) with both regimens starting right after tumor cell inoculation. Finally, ten rats were treated with zoledronic acid for 2 weeks before tumor cell inoculation (60 μg/kg/week sc × 2). The antiosteolytic effect of zoledronic acid was high as shown by inhibition of osteolytic growth. Addition of the anti-BSPII IgY further decreased the incidence of femoral osteolytic lesions (40% reduction), indicating remineralization, and reduced periosteal defects of cortical bone (20% reduction). These observations favor using the IgY-antibody in addition to zoledronic acid in order to stimulate osteoblast-induced remineralization.     

Induction of apoptosis by A3 adenosine receptor agonist N6-(3-iodobenzyl)-adenosine-5′-N-methylcarboxamide in human leukaemia cells: a possible involvement of intracellular mechanism

Aim: The sensitivity of cancer cells which exhibit multi-drug resistance phenotype to A3 adenosine receptor (A3AR) agonist N⁶-(3-iodobenzyl)-adenosine-5′-N-methylcarboxamide (IB-MECA) was studied. Methods: To establish direct relationship between P-glycoprotein (P-gp, ABCB1 and MDR1) expression and IB-MECA induced cell death, a straightforward method for precise estimation of intracellular level of this A3AR agonist was developed. Results: We subjected three human leukaemia cell lines HL-60, K562 and K562/HHT to treatment with micromolar concentrations of IB-MECA. Although all cell lines used expressed A3AR, there was a large difference in their sensitivity to IB-MECA. While HL-60 and K562 cells were almost equally sensitive, the K562/HHT cells, which exhibit a multi-drug resistance phenotype because of overexpression of P-gp, were significantly more resistant. We found that the intracellular level of IB-MECA in K562/HHT cells was approx. 10 times lower than those in HL-60 or K562 cells. Inhibitors of P-gp, including cyclosporine A (CsA) and verapamil (Vpa), increased the intracellular level of IB-MECA and reversed the resistance of K562/HHT cells to this drug. Accordingly, shRNA-mediated down-regulation of P-gp significantly increased the intracellular level of IB-MECA in K562/HHT cells which simultaneously exhibited reduced resistance to this A3AR agonist. In addition, an in vitro enzyme-based assay provided evidence that IB-MECA might serve as a substrate for P-gp. Conclusion: Our results suggest that P-gp overexpression prevents cells from IB-MECA induced apoptosis despite the A3AR expression. Pro-apoptotic effect of IB-MECA seemed to strongly depend on its intracellular accumulation rather than on its interaction with A3AR.     

Positive effect on bone fusion by the combination of platelet-rich plasma and a gelatin β-tricalcium phosphate sponge: a study using a posterolateral fusion model of lumbar vertebrae in rats

We developed a novel method for bone fusion by combining platelet-rich plasma (PRP) and a gelatin β-tricalcium phosphate (β-TCP) sponge. The PRP is an autologous concentration of platelets that includes several growth factors. The gelatin β-TCP sponge comprises gelatin and β-TCP, thus enabling the sustained release of growth factors and osteoconduction. To evaluate this method, we generated a posterolateral fusion model of lumbar vertebrae in rats and divided it into five groups by implanting the following materials between transverse processes of vertebrae, (1) the gelatin β-TCP sponge with PRP (PRP sponge), (2) the gelatin β-TCP sponge with platelet-poor plasma, (3) gelatin hydrogel with PRP, (4) autologous iliac bone (autograft), and (5) no material was implanted as a control. The assessment of bone fusion by a radiographic assessment, a biomechanical test, microcomputed tomography, and histological evaluations demonstrated that there were no significant differences between the PRP sponge and the autograft groups regarding the osteogenic effect. Subsequent examinations revealed that no significant differences existed between the PRP sponge and the autograft groups in either biomechanical stiffness or the bone volume over time; whereas the radiographic and histological composition underwent similar changes in the fusion process. These results indicate that the PRP sponge could, therefore, be potentially useful as an attractive and less invasive method for bone fusion.