Relationship between estrogen receptor concentration and cytomorphometry in breast cancer

This study aimed to evaluate the relationship between estrogen receptor (ER) concentration and cytomorphometric parameters in breast carcinoma. Primary breast cancer specimens were both imprinted on cytologic slides and submitted to ER determination with a dextran-coated charcoal method. Patients were stratified by ER levels ranging from 0 to greater than or equal to 200 fmol/mg protein. Measurements were performed on cytologic imprint technique smears in five cases from each ER strata, using a computer-assisted tracing device. Nuclear, cytoplasmic, and cellular parameters were measured on 50 cells per case. The cytometric findings were correlated with ER concentrations. A statistically significant correlation between decreasing area (P = 0.011), perimeter (P = 0.015), maximum diameter (P = 0.034), minimum diameter (P = 0.008), and volume (P = 0.01) of nuclei and increasing ER levels was found. With regard to whole cells, the following parameters significantly decreased versus increasing ER levels: area (P = 0.038), perimeter (P = 0.046), minimum diameter (P = 0.011), and volume (P = 0.044). A statistically significant relationship between the decreasing cytoplasmic perimeter (P = 0.025), i.e., nuclear plus cellular perimeter, and increasing ER content was found. Cytoplasmic area and nuclear to cytoplasmic ratio (N/C) did not correlate with the amount of ER. In all classes of different ER concentration, cells and nuclei displayed a regular shape closer to a circle than to an ellipse. The results of the current investigation indicate that tumors with higher ER concentration are composed of smaller cells with smaller nuclei than are tumors with lower ER content.     

Morphologic, histochemical, and functional analysis of platelet-rich plasma activity on skeletal cultured cells

BACKGROUND: Platelet-rich plasma (PRP) is a medium containing concentrated amounts of growth factors in a form that is easy to handle in regenerative sites. The aim of this study was to assess the effect of PRP on the differentiation of cultured skeletal cells and the capability of PRP to induce the production of some osteogenesis-related molecules and mineralization.
STUDY DESIGN AND METHODS: Flow cytometry (cellular antigens), real-time quantitative polymerase chain reaction (RT-qPCR; bone morphogenetic protein [BMP] messengers), alkaline phosphatase (ALP; osteogenic expression), and calcification analyses were performed on 24- and 48-hour human bone cells (HBCs) and 143B and SaOS-2 (osteosarcoma) cell cultures to study the effect of PRP on proliferation and differentiation of skeletal cultured cells. PRP was added using different protocols since no studies are available on bone cultures treated in the long term with PRP.
RESULTS: Flow cytometry showed PRP induction toward a nonhemopoietic lineage in HBCs; RT-qPCR showed enhanced mRNA encoding for BMP2 in HBCs, BMP6 and BMP7 in 143B cultures, and BMP2 and BMP7 in SaOS-2 cultures. Better ALP and calcification results were obtained in SaOS-2 cultures when PRP was added more frequently at shorter intervals while poor results were obtained after single PRP addition.
CONCLUSIONS: The results highlight induction of bone cell proliferation and differentiation by PRP. Since repeated administration of PRP is needed to achieve the best results, an almost continuous delivery system of PRP, or better a controlled release of growth and differentiation factors, using biomaterials might provide increased performance at bone regeneration sites.     

Treatment of cerebral ischemia with melanocortins acting at MC4 receptors induces marked neurogenesis and long-lasting functional recovery

Melanocortins produce neuroprotection against ischemic stroke with subsequent long-lasting functional recovery, through melanocortin MC(4) receptor activation. Here we investigated whether the long-lasting beneficial effect of melanocortins in stroke conditions is associated with a stimulation of neurogenesis. Gerbils were subjected to transient global cerebral ischemia by occluding both common carotid arteries for 10 min; then, they were prepared for 5-bromo-2'-deoxyuridine (BrdU) labeling of proliferating cells. Delayed treatment (up to 9 h after the ischemic injury) for 11 days with the melanocortin analog [Nle(4),D-Phe(7)]α-melanocyte-stimulating hormone (NDP-α-MSH) improved learning and memory throughout the 50-day observation period. Immunohistochemical examination of the hippocampus on day 50 showed, in the dentate gyrus, an elevated number of BrdU immunoreactive cells colocalized with NeuN (used as indicator of mature neurons) and Zif268 (used as indicator of functionally integrated neurons). Retrospective analysis during the early stage of neural stem/progenitor cell development (days 3 and 4 after stroke) showed, in NDP-α-MSH-treated gerbils, a high degree of daily cell proliferation and revealed that NDP-α-MSH favorably affects Wnt-3A signaling pathways and doublecortin expression. Pharmacologic blockade of MC(4) receptors prevented all effects of NDP-α-MSH. These data indicate that treatment of cerebral ischemia with MC(4) receptor agonists induces, with a broad window of therapeutic opportunity, long-lasting functional recovery associated with a large number of mature and likely functional newborn neurons in brain injured areas. Our findings reveal previously undescribed effects of melanocortins which might have major clinical implications.     

Selective melanocortin MC4 receptor agonists reverse haemorrhagic shock and prevent multiple organ damage

BACKGROUND AND PURPOSE: In circulatory shock, melanocortins have life-saving effects likely to be mediated by MC4 receptors. To gain direct insight into the role of melanocortin MC4 receptors in haemorrhagic shock, we investigated the effects of two novel selective MC4 receptor agonists. EXPERIMENTAL APPROACH: Severe haemorrhagic shock was produced in rats under general anaesthesia. Rats were then treated with either the non-selective agonist [Nle4, D-Phe7]-melanocyte-stimulating hormone (NDP--MSH) or with the selective MC4 agonists RO27-3225 and PG-931. Cardiovascular and respiratory functions were continuously monitored for 2 h; survival rate was recorded up to 24 h. Free radicals in blood were measured using electron spin resonance spectrometry; tissue damage was evaluated histologically 25 min or 24 h after treatment. Key results: All shocked rats treated with saline died within 30-35 min. Treatment with NDP--MSH, RO27-3225 and PG-931 produced a dose-dependent (13-108 nmol kg-1 i.v.) restoration of cardiovascular and respiratory functions, and improved survival. The three melanocortin agonists also markedly reduced circulating free radicals relative to saline-treated shocked rats. All these effects were prevented by i.p. pretreatment with the selective MC4 receptor antagonist HS024. Moreover, treatment with RO27-3225 prevented morphological and immunocytochemical changes in heart, lung, liver, and kidney, at both early (25 min) and late (24 h) intervals. Conclusions and Implications: Stimulation of MC4 receptors reversed haemorrhagic shock, reduced multiple organ damage and improved survival. Our findings suggest that selective MC4 receptor agonists could have a protective role against multiple organ failure following circulatory shock.     

Effects of COX1-2/5-LOX blockade in Alzheimer transgenic 3xTg-AD mice

Objective and design

Alzheimer’s disease (AD) is associated with amyloid plaques (Aβ) and hyperphosphorylated tau protein tangles in the brain. We investigated the possible neuroprotective role of flavocoxid, a dual inhibitor of cyclooxygenases-1/2 (COX-1/2) and 5-Lipoxygenase (5-LOX), in triple-transgenic (3xTg-AD) mice.

Subjects

Mice were 3 months at the beginning of the study.

Treatment

Animals received once daily for 3-month saline solution or flavocoxid (20 mg/kg/ip).

Methods

Morris water maze was used to assess learning and memory. Histology was performed to evidence Aβ plaques and neuronal loss, while inflammatory proteins were determined by western blot analysis.

Results

Saline-treated 3xTg-AD mice showed an impairment in spatial learning and memory (assessed at 6 months of age), and increased expression of inflammatory and apoptotic molecules. Treatment of 3xTg-AD mice with flavocoxid reduced: (1) learning and memory loss; (2) the increased eicosanoid production and the phosphorylation level of amyloid precursor protein (APP-pThr668), Aβ 1–42, p-tau (pThr181), pERK, and the activation of the NLRP3 inflammasome; (3) Aβ plaques; and (4) neuronal loss, compared to saline-treated animals.

Conclusions

Pharmacological blockade of both COX-1/2 and 5-LOX was able to counteract the progression of AD by targeting pathophysiological mechanisms up- and downstream of Aβ and tau.

     

PLGA microspheres for oral osteopenia treatment: preliminary "in vitro"/"in vivo" evaluation

The aim of this work was to prepare and to evaluate "in vitro"/"in vivo" microspheres based on poly(D,L-lactide-co-glycolide) copolymers containing ipriflavone, for the local treatment of oral bone loss. The first objective was the preparation and "in vitro" characterization of ipriflavone loaded microspheres, by emulsion/solvent evaporation method. Process parameters such as drug:polymer weight ratio, and molecular weight of copolymers, were also investigated. The second objective was to elaborate a suitable animal model of mandibular osteoporosis, to evaluate the efficacy of these microparticulate drug delivery systems. "In vivo" experiments were carried out on female rats, in which oral osteopenia was induced by gonadectomy and molar avulsion. Morphometric analysis of mandibular segment were carried out to quantify the development of oral osteopenia and the efficacy of drug loaded microspheres. Results showed that ipriflavone loaded PLGA microspheres can be successfully obtained with good "in vitro" characteristics, utilizing the emulsification/solvent evaporation method. "In vivo" experiments revealed that local administration of microspheres produced only mild inflammation on the injection site. Morphometric analyses showed, at the level of the third molar, a slight increase in spongy and total bone mass on rat jaw treated with microspheres with respect to control. Control animals exhibited a scarce degree of osteopenia demonstrating that this animal model is not suitable for this purpose.     

Effect of late treatment with gamma-hydroxybutyrate on the histological and behavioral consequences of transient brain ischemia in the rat

It has been previously described that gamma-hydroxybutyrate (GHB) provides significant protection against transient global cerebral ischemia in the rat (four vessel occlusion model), when given 30 min before or 10 min after artery occlusion. Here, we show that in the same rat model, significant protection can also be obtained when treatment is started 2 h after the ischemic episode. In saline-treated animals, 30 min of global ischemia followed by reperfusion caused a massive loss of neurons in the hippocampal CA1 subfield (examined 63 days after the ischemic episode), and an impairment of sensory-motor performance (tested on the 51st and 63rd days after ischemia) and of spatial learning and memory (evaluated starting 46 days after the ischemic episode). Treatment with GHB--300 mg/kg intraperitoneally (i.p.) 2 h after the ischemia-reperfusion episode, followed by 100 mg/kg i.p. twice daily for the following 10 days--afforded a highly significant protection, against both histological damage and sensory-motor and learning-memory impairments. These data further suggest the possible therapeutic effectiveness of GHB in brain ischemia, and indicate that the underlying mechanism of action involves non-immediate steps of the ischemia-induced cascade of events.     

Both early and delayed treatment with melanocortin 4 receptor-stimulating melanocortins produces neuroprotection in cerebral ischemia

Ischemic stroke is one of the main causes of death and disability. We investigated whether melanocortin peptides, which have protective effects in severe hypoxic conditions, also produce neuroprotection in a gerbil model of ischemic stroke. A 10-min period of global cerebral ischemia, induced by occluding both common carotid arteries, caused impairment in spatial learning and memory that was associated with activation of inflammatory and apoptotic pathways, including severe DNA damage and delayed neuronal death, in the hippocampus. Treatment with nanomolar doses of the melanocortin analog [Nle4, D-Phe7] alpha-MSH [which activates the melanocortin receptor subtypes (MC) mainly expressed in central nervous system, namely MC3 and MC4] modulated the inflammatory and apoptotic cascades and reduced hippocampus injuries even when delayed up to 9 h after ischemia, with consequent dose-dependent improvement in subsequent functional recovery. The selective MC3 receptor agonist gamma2-MSH had no protective effects. Pharmacological blockade of MC4 receptors prevented the neuroprotective effects of [Nle4, D-Phe7] alpha-MSH and worsened some ischemia outcomes. Together, our findings suggest that MC4 receptor-stimulating melanocortins might provide potential to develop a class of drugs with a broad treatment window for a novel approach to neuroprotection in ischemic stroke.