Immunohistochemical Detection Of Ki-67 Research Articles

Comparative study on the degradation rate and regulatory effects of two resorbable collagen membranes during the in vivo implantation

Objective: To explore the differences in the performance and tissue repair promotion effects of small intestinal submucosa membrane (SIS membrane) and Bio-Gide resorbable collagen membrane (Bio-Gide membrane) by performing the subcutaneous implantation models in mice. Methods: For in vivo studies, we stablished membrane implantation models using 6-8 week-old male C57BL/6 mice. The degradation rates were explored through HE staining analysis at different time points (1, 3, 5, 7, 14 and 28 d, 3 mice/group/time point). The influences of the two membranes on local macrophages and neovasculum were evaluated by immunofluorescence detection of F4/80 and CD31, and the mobilization effects of the two membranes on local stem cells were evaluated by immunohistochemical detection of Ki67 and CD146. For in vitro studies, mice periodontal ligament stem cells (mPDLSCs) were co-cultured with these two membrane materials, and the cell morphologies were observed by scanning electron microscopy. In addition, the gene expressions of Ki67, Cxcl1, Ccl1, Tnfa were investigated by real-time fluorescence quantitative PCR (RT-qPCR). Results: The results of in vivo studies showed that by day 28, there was no significant difference in degradation rate between these two membrane materials [SIS degradation rate: (16.84±4.00) %, Bio-Gide degradation rate: (24.07±3.97) %, P=0.090], illustrating that both of them could maintain the barrier effects for more than one month. In addition, there was no significant difference in the infiltration number of local F4/80 positive macrophages between these two groups by the day 3 after implantation [SIS: (20.67±5.69) cells/visual field, Bio-Gide: (25.33±2.52) cells/visual field, P=0.292]. However, compared with the Bio-Gide membrane, SIS membrane significantly promoted local CD31+vascular regeneration [SIS: (4.67±1.15) cells/visual field, Bio-Gide: (1.00±1.00) cells/visual field, P=0.015] and CD146+stem cell recruitment [SIS: (22.33±4.16) cells/visual field, Bio-Gide: (11.33±2.52) cells/visual field, P=0.025]. The RT-qPCR results also showed that SIS membrane promoted the gene expression of Cxcl1 (SIS vs Bio-Gide P<0.001) in mPDLSCs, but had no effect on the gene expression of Tnfa (SIS vs Bio-Gide P=0.885). Conclusions: SIS membrane showed a similar degradation rate compared with Bio-Gide membrane, and there was no significant difference in the effects of these two membranes on local inflammation or macrophages. Therefore, both of these membranes could meet the barrier effects required by guided tissue regeneration.

Hepatocyte proliferation activity in untreated rats, measured by immunohistochemical detection of Ki-67: The effect of age on the repeated-dose liver micronucleus assay.

The repeated-dose liver micronucleus (RDLMN) assay is a widely accepted method for detecting genotoxic substances. We investigated the effect of animal age on this assay. Proliferation activity in the liver tissue of untreated rats at age=3.5, 6, 8, 10, or 12 weeks was measured via immunohistochemical expression of Ki-67 protein. The percentage of Ki-67-positive hepatocytes decreased markedly with age, reaching very low levels after 10 weeks, indicating decline with age of proliferative capacities in the liver. We calculated the area under the curve (AUC) of the approximate curve generated from the percentage of Ki-67-positive cells, to estimate the hepatocyte proliferation activity over the dosing period in the two regimens of the 4-week RDLMN assay: dosing initiated at age=6 or 8 weeks. Hepatocyte proliferation activity of the former regimen was approximately double that of the latter. We also calculated the AUC for the juvenile-rat method, in which rats are treated for two days at age=3.5 weeks. The AUC calculated for that method was approximately half of that for the 4-week repeated-dosing regimen initiated at 6 weeks of age. These findings suggest that the 4-week RDLMN assay with dosing initiated at age=6 weeks could be approximately twice as sensitive as the other two methods.

Effect of Sevoflurane on the Proliferation of A549 Lung Cancer Cells

Background and Objectives: Sevoflurane has opposing effects on cancer progression, depending on its concentration and the cancer type. This study investigated the effects of sevoflurane on the proliferation of A549 lung cancer cells. Materials and Methods: In vitro, the number of A549 cells exposed to different concentrations of sevoflurane was counted. The size and weight of tumors from a xenograft mouse model exposed to air or sevoflurane were measured in vivo experiments. Additionally, hematoxylin and eosin staining and immunohistochemical detection of Ki-67 in the harvested tumor tissues were performed. Results: A total of 72 culture dishes were used and 24 dishes were assigned to each group: Air group; 2% Sevo group (air + 2% sevoflurane); and 4% Sevo group (air + 4% sevoflurane). The number of A549 cells in the 2% Sevo group was less than that in the Air and 4% Sevo groups (Air: 7.9 ± 0.5; 0.5, 2% Sevo: 6.8 ± 0.4, 4% Sevo: 8.1 ± 0.3; p = 0.000). The tumor size was not significantly different between the two groups (Air: 1.5 ± 0.7, 2% Sevo: 2.4 ± 1.9; p = 0.380). Conclusions: The in vitro data showed that sevoflurane inhibited the proliferation of A549 lung cancer cells in a concentration-specific manner. However, the in vivo data showed no correlation between sevoflurane exposure and A549 cell proliferation. Thus, further research is required to understand fully the effects of sevoflurane on cancer progression and to reconcile differences between the in vitro and in vivo experimental results.

Platelet-rich plasma and ovarian quiescence: a bovine in vitro model for regeneration of the ovary.

Ovarian quiescence can be due to hormonal deficiency usually caused by apoptosis of granulosa cells responsible for oestrogen synthesis. This study evaluated the regenerative effect of platelet rich plasma (PRP) on bovine in vitro models to understand its effect on granulosa cells. Quiescent and healthy ovarian sections were cultured in the presence/absence of PRP for 72h and, at different times (0, 24, 48 and 72h), hematoxylin-eosin and immunohistochemical detection of Ki-67 were performed. Additionally, granulosa cells collected from healthy bovine ovaries were stressed with 100ng/mL of lipopolysaccharide (LPS) in presence/absence of PRP and evaluated at 0, 4, 8 and 24h for apoptosis by acridine orange and propidium iodide staining. Enzyme-linked immunosorbent assay tests were performed to evaluate oestrogen (E2) and anti-Müllerian hormone (AMH) concentrations on cultures of ovarian slices and granulosa cells. In slides of quiescent ovaries treated with PRP, a marked and widespread positivity to Ki-67 was expressed by 40-60% of the follicular wall cells at 48h of culture. Levels of E2 and AMH were significantly higher compared to untreated quiescent samples reaching the levels of healthy control samples. PRP counteracted the LPS effect and apoptosis (at 24h, there were 93.44±3.51% live cells with LPS+PRP compared to 37±1.32% with LPS) and significantly increased concentrations of E2 and AMH. PRP can stimulate granulosa cell proliferation and counteract inflammatory processes in vitro . This treatment could improve the reproductive ability of quiescent females.

DIAGNOSTIC UTILITY OF IMMUNOHISTOCHEMICAL EXPRESSION OF KI-67, P63 AND AMACR IN PROSTATE INTRAEPITHELIAL NEOPLASIA

The aim: To determine malignant transformation and progression ability of high grade and low grade prostate intraepithelial neoplasia with the help of immunohistochemical method. Materials and methods: The results of examination of 93 patients with PIN (50 patients with high grade PIN and 43 patients with low grade PIN) were assessed comparatively using immunohistochemical markers. Semiquantitative method was used to evaluate !"-67, #63 and AMACR tissue expression with four grades from "+" to "++++" or from 1 to 4 points: '+' - low reaction, '++' - poor reaction, '+++' - moderate reaction and '++++" - intense reaction. Results: There were statistically significant differences in immunohistochemical expression rates between HGPIN and LGPIN. Patients with HGPIN had higher Ki-67 and AMACR expression rate and lower p63 expression rate than patients with LGPIN. Intense and moderate Ki-67 expression was detected in HGPIN more often, in 24 % and 11 % respectively. Low and moderate AMACR expression was determined in HGPIN more often, in 28 % and 5 % respectively. Low and not evident p63 expression was observed in HGPIN more often, in 36 % and 8 % respectively. Conclusions: HGPIN has common morphological peculiarities with prostate adenocarcinoma. Immunohistochemical detection of Ki-67, p63 and AMACR is aimed to differentiate among patients with PIN a group of high malignant transformation risk.

The Role Of Amino Acid Arginine And Nitric Oxide System In Implementing Cardioprotective Effect Of Non-Opioid Analogue Of Leu-Enkephalin In Newborn Albino Rats After Intrauterine Hypoxia

Objective ― to evaluate the role of the amino acid arginine in the structure of the non-opioid analogue of leu-enkephalin (NALE) and the involvement of the nitric oxide system in the implementation of its cardioprotective effect in newborn albino rats subjected to intrauterine hypoxia. Material and Methods ― Pregnant female rats were subjected daily to 4-hour hypobaric hypoxia (oxygen partial pressure – 65 mm Hg) on days 15-19 of their gestation. The 7-day-old offspring of hypoxified female rats were examined. The progeny of intact animals served the control. We studied body and heart weights; activity of proliferative processes and autophagy in the myocardium of subendocardial parts of the left ventricle, expressed via the immunohistochemical detection of Ki-67 and Beclin-1 proteins, respectively; karyometric and nucleolometric indicators of cardiomyocytes (CMC); intensity of free radical processes in the tissues of the heart by chemiluminescence parameters. Correction of post-hypoxic changes in newborn rats was carried out by intraperitoneal injection of two peptides (Phe–D-Ala–Gly–Phe–Leu–Arg – non-opioid analogue of leu-enkephalin, or NALE, and Phe–D-Ala–Gly–Phe–Leu–Gly – G peptide) daily from day 2 through day 6 of their lives at a dose of 100 μg/kg. To assess the involvement of the nitric oxide system in the implementation of the NALE effects, the NO synthase inhibitor – N-nitro-L-arginine methyl ester (L-NAME) was additionally administered at a dose of 50 mg/kg. Results ― Intrauterine hypoxia led to a decrease in body weights of 7-day-old animals, an increase in the number of CMC expressing the Beclin-1 protein, reduction in the size of CMC nuclei, activation of free radial oxidation, and a decrease in antiradical protection in the heart tissues. The administration of NALE to newborn animals, subjected to intrauterine hypoxia (IUH), normalized their body weight and size of the CMC nuclei, and partially corrected changes in Beclin-1 expression and in chemiluminescence parameters. In 7-day-old animals, subjected to IUH and neonatal administration of NALE and L-NAME, a lower body weight was observed than in the control. Against the background of nitric oxide blockade, the antioxidant effect of NALE diminished, but the corrective effect of NALE on the karyometric index and Beclin-1 expression remained. G peptide, which differs from NALE by the substitution of the C-terminal amino acid Arg for the amino acid Gly, exhibited a corrective effect similar to NALE on the consequences of IUH. Conclusion ― Administration of NALE and G peptides to newborn albino rats after IUH has a pronounced cardioprotective effect. The mechanisms of the NALE peptide effects are, in part, associated with the activation of the NOS-NO system. However, the affinity of this peptide for opioid-like receptors may be of greater importance.

Metformin and melatonin improve histopathological outcome of NMU-induced mammary tumors in rats

ObjectiveNumerous reports showed inhibition of carcinogenesis after metformin (MF) and melatonin (MEL) administration. However, most in vivo studies used standard diet type, with relatively low fat content. As increase in fat intake may have a considerable impact on malignant transformation, we evaluated the effects of these two substances in a model of mammary carcinogenesis in rats fed a high-fat diet (10%). MethodsMammary tumors were induced by N-methyl-N-nitrosourea (NMU) in female rats of sensitive Sprague-Dawley strain. MF was administered in a diet (0.2%), MEL was administered in drinking water (20 mg/L). The chemoprevention was initiated 12 days prior to tumor initiation, both substances were administered through the termination of the experiment on 16th week after carcinogen application. Analysis of basic parameters of tumor growth, histopathological profile, and serum IGF-1 level were performed together with immunohistochemical detection of Ki67 (proliferation marker) and caspase-3 and BCL-2 (apoptosis markers) in mammary cancer cells. ResultsAlthough neither tumor incidence nor frequency were changed after MF and/or MEL administration, MF and MEL decreased high-grade/low-grade (HG/LG) tumor ratio. MEL decreased proliferation in mammary cancer cells; positive correlations between histological grade and Ki67 expressions were found after single administration of both MF and MEL. Serum IGF-1 levels were reduced to the level of intact rats in all groups receiving chemoprevention. ConclusionsMF and MEL administration did not inhibit growth of NMU-induced mammary tumors in rats in a significant manner but both substances ameliorated tumor histopathological profile. Surprisingly, combined treatment had no such effect.

Aflatoxin induced renal toxicity in Albino rats and the ameliorative effect of green tea aqueous extract: Histological, Morphometric and Immuno-histochemical Study

Tea is the most common drink worldwide besides water. It is rich in antioxidant polyphenolic flavonoids. Further, it has anti-apoptotic, anti-inflammatory, and autoantigen-inhibitory properties. To evaluate the effects of oral administration of aqueous extract of green tea against experimental aflatoxicosis on the renal tissues of adult male albino rats. Twenty five adult male rats were included and they were divided equally into 5 groups: Group 1 (aflatoxin group): the rats received aflatoxins (2.5 mg/kg diet for 15 days), group 2 (aflatoxins and green tea aqueous extract group) for 15 days, group 3, the rats received aflatoxins for 15 days and aqueous extract of the green tea for 30 days, group 4 (green tea aqueous extract group) and group 5(control negative group). Detailed non-traditional histological and morphometrical analyses for renal tissues stained with Hematoxylin and Eosin, Masson's trichrome stain, Periodic Acid Schiff’s (PAS), Alizarin red S stains and immunohistochemical detection of Ki-67 were done. The main observed renal lesions in rats received aflatoxins were dilatation of the renal pelvis, increases in glomerular areas (GA), widening of Bowman's spaces and necrosis of the renal lining epithelium and proliferation of fibrous connective tissues. Morphometric analysis revealed the presence of significant difference between group I and the other experimental groups. Meanwhile, groups 2 and 3 revealed a significant improvement in the histological changes and decrease in most of parameters. The highest percentage of PAS positive reaction was significantly found in group 4. Quantitative analysis of positive reactions of Ki-67 revealed the highest positive immunohistochemical reactions were detected in group 1 which significantly different in groups 4 and 5. In conclusion, green tea has a protective effect against the adverse effects of aflatoxins on renal tissues evidenced by improvement of histopathological alterations and regulation of Ki-67 expressions.&#x0D;

Metformin and melatonin inhibit DMBA-induced mammary tumorigenesis in rats fed a high-fat diet.

The data from in-vitro and in-vivo studies show that both peroral antidiabetic metformin (MF) and pineal hormone melatonin (MT) inhibit the growth of many cancers, including breast cancer. However, most in-vivo studies used standard-type diet with low fat content. Therefore, in this study, we evaluated the chemopreventive effect of MF and MT in an in-vivo model of breast cancer in rats on a high-fat diet (10% of total fat). Mammary carcinogenesis was induced by 7,12-dimethylbenz[a]anthracene (DMBA) in female Sprague-Dawley rats. Chemoprevention with MF (administered in a diet, 0.2%) and MT (administered in tap water, 20 mg/l) was induced 20 days before the carcinogen administration through the termination of the experiment (14 weeks after carcinogen administration). Tumor growth parameters were analyzed together with histopathological examination and immunohistochemical detection of KI67 (proliferation marker), caspase-3, BAX, BCL-2 (apoptosis markers), and CD24 and CD44 (cancer stem cell markers) in mammary tumor samples. The combination of chemopreventive agents decreased tumor incidence by 29%. Cumulative tumor volume was lower in all groups treated with chemoprevention. Histopathology did not show significant changes in high-grade/low-grade tumor ratio. Immunohistochemistry showed increased expression of BAX in the combination group, and caspase-3 expression increased in both MT and combination groups. MT, and particularly the MF and MT combination, inhibited DMBA-induced mammary tumor growth in rats by apoptosis stimulation in cancer cells. Our results indicate that MT supplements in patients treated with MF may have a considerable effect on the incidence of breast cancer.

Periovulatory follicular fluid levels of estradiol trigger inflammatory and DNA damage responses in oviduct epithelial cells.

ObjectiveOvarian steroid hormones (mainly E2 and P4) regulate oviduct physiology. Serum-E2 acts on the oviduct epithelium from the basolateral cell compartment. Upon ovulation, the apical compartment of the oviduct epithelium is temporarily exposed to follicular fluid, which contains much higher levels of E2 than serum. The aim of this study was to evaluate the effects of human periovulatory follicular fluid levels of E2 on oviduct epithelial cells using two porcine in vitro models.MethodsA cell line derived from the porcine oviductal epithelium (CCLV-RIE270) was characterized (lineage markers, proliferation characteristics and transformation status). Primary porcine oviduct epithelial cells (POEC) were cultured in air-liquid interface and differentiation was assessed histologically. Both cultures were exposed to E2 (10 ng/ml and 200 ng/ml). Proliferation of CCLV-RIE270 and POEC was determined by real-time impedance monitoring and immunohistochemical detection of Ki67. Furthermore, marker gene expression for DNA damage response (DDR) and inflammation was quantified.ResultsCCLV-RIE270 was not transformed and exhibited properties of secretory oviduct epithelial cells. Periovulatory follicular fluid levels of E2 (200 ng/ml) upregulated the expression of inflammatory genes in CCLV-RIE270 but not in POEC (except for IL8). Expression of DDR genes was elevated in both models. A significant increase in cell proliferation could not be detected in response to E2.ConclusionsCCLV-RIE270 and POEC are complementary models to evaluate the consequences of oviduct exposure to follicular fluid components. Single administration of periovulatory follicular fluid E2 levels trigger inflammatory and DNA damage responses, but not proliferation in oviduct epithelial cells.

Abstract P1-12-06: UCBG intergroup: 3-years efficacy results of the Unicancer-PACS08 trial including poor prognosis patients treated with docetaxel or ixabepilone in adjuvant setting

Abstract Background: Ixabepilone, an epothilone B analog, has demonstrated single-agent activity in metastatic and neoadjuvant settings. The PACS08 trial aimed to compare adjuvant FEC100-Docetaxel regimen to FEC100-Ixabepilone in poor prognosis early breast cancer (BC) composed of patients presenting with triple-negative (TN) [i.e. estrogen receptor (ER)-/progesterone receptor (PR)-/HER2-] or ER+/PR-/HER2- tumor, which are subgroups significantly associated with worse prognosis. Patients and methods: Between 2007 and 2010, 762 patients with unilateral TNBC (n=592, 78%) or node-positive ER+/PR-/HER2- BC (n=170, 22%) were enrolled. Recruitment was interrupted due to BMS decision to stop ixabepilone development in adjuvant setting. Main inclusion criteria were: age&amp;lt;70 years, normal cardiac, hepatic, haematological and renal functions. Arm A: pts received 3 cycles of FEC100 (F and C, each at 500 mg/m2, E 100 mg/m2, every 3 weeks) followed by 3 cycles of Docetaxel (100 mg/m2 every 3 weeks); Arm B, Ixabepilone 40 mg/m2 replaced Docetaxel. Radiotherapy was mandatory after conservative surgery and endocrine therapy was given to ER+ pts. ER, PR and HER2 status were validated by a central pathology review on 754 cases and Immunohistochemical detection of Ki67, EGFR, cytokeratins (CK) 5/6 and 14, was performed on tissue microarray (TMA). Results: Main pts characteristics were well balanced between the 2 arms. As of September 2014, the median follow-up was 36 months. The safety profile indicates that Docetaxel is more often associated to significant haematological toxicities whereas both neurotoxicities and haematological toxicities are reported in Ixabepilone arm. Log-Rank tests indicate no difference between two arms in terms of both DFS and OS (HR=1.2, 95%CI (0.864-1.728), p=0.256 and HR=1.8, 95%CI (0.751-1.855), p=0.473, respectively). Pathological analysis of the PACS08 collection showed that TNBC displayed significantly higher proliferative activity as shown by mitotic count and Ki67 index (p&amp;lt;0.001). As compared to ER+/PR-/HER2- subgroup, TNBC showed distinct characteristics, and displayed a so-called basal-like phenotype in 80%. Further efficacy analyses are ongoing in order to study whether chemotherapy may have better prognosis according to pathological characteristics including tumor lymphocytic infiltrate. These additional data will be available for the SABCS2015 meeting. Conclusions: Our results indicate that Ixabepilone doesn't show higher efficiency compared to Docetaxel in adjuvant setting in poor prognosis early breast cancer. We have an unusual biological collection associated to our clinical data which will allow us to correlate efficacy data to breast cancer subgroups. Other several translational researches are still ongoing. Citation Format: Campone M, Lacroix-Triki M, Roca L, Spielmann M, Wildiers H, Cottu P, Kerbrat P, Levy C, Mayer F, Bachelot T, Wiston T, Eymard J-C, Uwer L, Machiels J-P, Verhoeven D, Jaubert D, Facchini T, Orfeuvre H, Canon J-L, Asselain B, Lemonnier J, Roché H. UCBG intergroup: 3-years efficacy results of the Unicancer-PACS08 trial including poor prognosis patients treated with docetaxel or ixabepilone in adjuvant setting. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-12-06.

Роль иммуногистохимического исследования при диагностике опухолевых поражений шейки матки

The analysis of literature data about the use of immunohistochemical markers for the diagnosis and differential diagnosis of cervical lesions. The most significant assistance in identifying and determining the degree of cervical intraepithelial neoplasia has immunohistochemical detection of Ki-67 and p16. Shows the changes of p63, topoisomerase, C-erbB-2, cyclin D1 at the cervical intraepithelial neoplasia of different degrees of severity and cervical cancer. It is concluded that the immunohistochemical research is legitimate considered as an effective method for histopathological studies. It allows to carry out an objective diagnosis of neoplastic lesions of the cervix, to carry out their differential diagnosis with reactive and tumor-like changes, as well as to determine the prognosis of the disease.

Effects of early and late treatment with soy isoflavones in the mammary gland of ovariectomized rats

ABSTRACTIntroduction Soy isoflavones have been shown to be an alternative to hormone therapy at menopause, without causing side-effects such as breast cancer. However, the effects of early and late treatment with isoflavones on the mammary gland remain controversial.Objective To investigate the effects of early and late treatment with soy isoflavones on the mammary gland of ovariectomized rats.Methods Thirty 3-month-old rats were ovariectomized and divided equally into groups: Control, treated with vehicle solution; or with 150 mg/kg/body weight of isoflavones by gavage; or subcutaneously treated with 10 μg/kg/body weight with 17β-estradiol. Treatments started 3 days (early treatment) or 30 days (late treatment) after ovariectomy and lasted for 30 consecutive days. Thereafter, the animals were euthanized and the mammary glands were removed and processed for paraffin embedding. Sections were stained with hematoxylin and eosin for histomorphometry or subjected to immunohistochemical detection of Ki-67 and VEGF-A.Results The ductal, lobular and total epithelial fractions were similar between controls and the early/late isoflavone groups, but they were significantly higher in the groups treated with estradiol. In both epithelial and stromal regions, the immunoreactivity of VEGF-A and the percentage of Ki-67-positive cells were significantly higher in the groups treated with estradiol, while they were similar in the early/late isoflavone groups and control groups.Conclusion Our results indicate that early and late treatment with soy isoflavones at the dose of 150 mg/kg/body weight does not show proliferative and angiogenic effects on the mammary gland of ovariectomized rats.

Evaluation of the antiulcer potential of Punica granatum (pomegranate) peel extract on indomethacin-induced duodenal ulcer

Introduction: Duodenal ulcer is a serious public health problem that affects approximately 10% of the global population and is two to three times more prevalent than gastric ulcer. Naturally occurring medications for duodenal ulcer include the use of pomegranate rind powder. This traditional medication has been used in Saudi Arabia for many years. Objectives: To investigate the capacity of pomegranate peel extract in healing and preventing duodenal ulceration in rats. Materials and methods: Experimental animals were divided into four groups. Group I was used as a control. In Group II (ulcer group) duodenal ulcer was induced by indomethacin administration using a single oral dose of 25mg/kg. In Group III (prevention group), 2 weeks before ulcer induction, rats were treated orally with pomegranate peel extract or ranitidine. In Group IV (treatment group), after ulcer induction, rats were treated with pomegranate peel extract or ranitidine for 2 weeks. The duodenum was subjected to light microscopic studies, immunohistochemical detection of Ki67 as a proliferative marker and biochemical studies (measuring the parameter of lipid peroxidation). Statistical analyses were carried out for determination of the significance of changes in Ki67 proliferation index. Results: In the ulcer group, the levels of malondialdehyde were significantly increased while the reduced glutathione activity was lowered in duodenal mucosa compared with the control group. Apparent disturbed villous to crypt ratio with necrosis and cellular lysis at the tip of the villi and marked mononuclear cellular infiltration were observed in the ulcer group. There was preservation of the crypt to villous ratio, an increase in goblet cells and decreased levels of malondialdehyde in the protection and treatment groups. The number of Ki67 immunostained cells was also increased in these groups. Conclusions: The use of pomegranate rind powder gave good results in the treatment, and perhaps also prevention, of duodenal ulceration in rats.

Age-related changes in human periosteal derived stem cells: a matter for effective bone regeneration strategies

Possible age-related changes in Mesenchymal Stem Cells (MSCs) are of great inter- est in view of their use for regenerative medicine approaches also in the elderly. Con- sidering the primary role of periosteum in bone biology and to acquire data for a cell- based therapy stimulating graft osseointegration, we tried to identify specific aging markers or pattern of expression in human periosteal precursor cells (PDPCs). To this aim periosteal tissue was obtained from differently aged healthy subjects, gender matched with a mean age of 16, 28, 63 and 92 years. Immunohistochemical detection of Ki67 and p53, Nitric Oxide (NO) production and qRT- PCR of a selected gene pan- el for early osteoblastic differentiation (bmp2 and runx2) and bone remodelling (IL-6, RANKL and OPG) were evaluated. Our data evidenced that both Ki67 and p53 rep- resent striking markers of cell-cycle arrest in these cells and that their expression cor- relates with NO production. In addition, age affects genes involved in bone remodel- ling, with a significant increase in IL-6 mRNA expression as well as in RANKL/OPG ratio. As far as NO release is concerned, our data showed higher levels in PDPCs iso- lated from the elderly and a good correlation with the immunohistochemical analysis Moreover, mathematical modelling, in silico simulations and biochemical experiments were combined to investigate about possible underlying quantitative correlations. A clear one-term exponential relationship emerged from a comparison of involved marker trends against age of donors concerning measured NO concentration / Ki67 ratio. This analytical approach confirmed Ki67 as a senescence marker to be focused on. We believe that this study, taking into account age-related changes in human PDPCs, opens up new regenerative medicine strategies for aged bone and/or bone metabolic diseases.

Pathohistological and immunohistochemical parameters significant for prognosis and therapy of tumours in cats and dogs

The development of tumours implies a disproportion between proliferation, growth and differentiation, which is accompanied by significant genotypic and phenotypic variations in the diseased. The scenario of multiple oncogenic effects on the inhibition of cell growth, apoptosis and maturation, on the stimulation of proliferation, on cell migration and tissue invasion, is responsible for the occurrence of tumours both in humans and in animals. The qualitative and quantitative determination of biological prognostic factors in the tissue of affected cats and dogs directly indicates a possible tumour metastasizing, and consequently also the prognosis of neoplastic disease. Monitoring of clinical and biological prognostic factors is important for therapy and prediction of tumours in humans and animals. The determination of the proliferative potential of tumour cells by immunohistochemical detection of Ki-67 and PCNA is used in grading certain tumours in cats and dogs. It is also possible immunohistochemically to prove factors of angiogenesis and numerous activating and inhibiting proteins in the tumour tissue. In addition to other histological, clinical and biological parameters, they are significant for the further prognosis and therapy of neoplastic diseases.

Polymeric micelles encapsulating fisetin improve the therapeutic effect in colon cancer.

The natural flavonoid fisetin (3,3',4',7-tetrahydroxyflavone) was discovered to possess antitumor activity, revealing its potential value in future chemotherapy. However, its poor water solubility makes it difficult for intravenous administration. In this study, the monomethyl poly(ethylene glycol)-poly(ε-caprolactone) (MPEG-PCL) copolymer was applied to prepare nanoassemblies of fisetin by a self-assembly procedure. The prepared fisetin micelles gained a mean particle size of 22 ± 3 nm, polydisperse index of 0.163 ± 0.032, drug loading of 9.88 ± 0.14%, and encapsulation efficiency of 98.53 ± 0.02%. Compared with free fisetin, fisetin micelles demonstrated a sustained and prolonged in vitro release behavior, as well as enhanced cytotoxicity, cellular uptake, and fisetin-induced apoptosis in CT26 cells. As for in vivo studies, fisetin micelles were more competent for suppressing tumor growth and prolonging survival time than free fisetin in the subcutaneous CT26 tumor model. Furthermore, histological analysis, terminal deoxynucleotidyl transferase-mediated nick-end labeling assay, immunohistochemical detection of Ki-67, and microvessel density detection were conducted, demonstrating that fisetin micelles gained increased tumor apoptosis induction, proliferation suppression, and antiangiogenesis activities. In conclusion, we have successfully produced a MPEG-PCL-based nanocarrier encapsulating fisetin with enhanced antitumor activity.

Human Periosteal Derived Stem Cell Potential: The Impact of age.

There is a great deal of interest in the understanding of possible age-related changes in Mesenchymal Stem Cells in view of their use for regenerative medicine applications. Given to the outmost standing of periosteum in bone biology and to probe data for a cell-based therapy promoting graft osseointegration in the elderly, we tried to identify specific aging markers or pattern of expression in human periosteal precursor cells. Immunohistochemical detection of Ki67 and p53, Nitric Oxide production and qRT- PCR of a selected gene panel for osteoblastic differentiation, bone remodeling and stemness were evaluated. We confirmed that both Ki67 and p53 are noteworthy indicators of senescence in human periosteal precursor cells and their expression significantly correlate with cell NO production. Moreover, cell age affects genes involved in bone remodeling, with a significant increase in interleukin-6 mRNA expression and receptor activator of nuclear factor kappa-B ligand/osteoprotegerin ratio. The analysis of mRNAs of genes involved in pluripotency regulation and self-renewal of stem cells, evidenced changes at least in part related to bone remodeling. We believe that this is the first study taking on age-related changes in human periosteal precursor cells, and paving the way toward new regenerative medicine strategies in bone aging and/or bone metabolic diseases.

Expression of cell cycle and apoptosis-related proteins in ameloblastoma and keratocystic odontogenic tumor

Tumors arising from epithelium of the odontogenic apparatus or from its derivatives or remnants exhibit considerable histologic variation and are classified into several benign and malignant entities. A high proliferative activity of the odontogenic epithelium in ameloblastoma (AM) and keratocystic odontogenic tumor (KCOT) has been demonstrated in some studies individually. However, very few previous studies have simultaneously evaluated cell proliferation and apoptotic indexes in AM and KCOT, comparing both lesions. The aim of this study was to assess and compare cell proliferation and apoptotic rates between these two tumors. Specimens of 15 solid AM and 15 KCOT were evaluated. The proliferation index (PI) was assessed by immunohistochemical detection of Ki-67 and the apoptotic index (AI) by methyl green-pyronin stain. KCOT presented a higher PI than AM (P < .05). No statistically significant difference was found in the AI between AM and KCOT. PI and AI were higher in the peripheral cells of AM and respectively in the suprabasal and superficial layers of KCOT. In conclusion, KCOT showed a higher cell proliferation than AM and the AI was similar between these tumors. These findings reinforce the classification of KCOT as an odontogenic tumor and should contribute to its aggressive clinical behavior.

Abstract P3-05-07: Poor prognosis early breast cancer: pathological characteristics of the Unicancer-PACS08 trial including patients treated with docetaxel or ixabepilone in adjuvant setting

Abstract Background: The PACS08 trial aimed to compare adjuvant FEC100-Docetaxel regimen to FEC100-Ixabepilone (Ixa) in poor prognosis early breast cancer (BC). The study population included BC patients presenting with triple-negative (TN) [i.e. estrogen receptor (ER)−/progesterone receptor (PR)−/HER2−] or ER+/PR−/HER2− tumor, which are subgroups significantly associated with worse prognosis. Central review was performed and detailed pathological characteristics of the cohort are reported herein. Patients and method: Between 2007 and 2010, 762 patients with unilateral TNBC (n = 592, 78%) or ER+/PR−/HER2− BC (n = 170, 22%) were enrolled. Recruitment was interrupted due to BMS decision to stop Ixa development in adjuvant setting. As defined by inclusion criteria, TNBC were either node+ or node-, and ER+/PR−/HER2− BC only node+. Following the validation of ER, PR and HER2 status on whole sections prior to inclusion, paraffin blocks (n = 754) were sent for central pathology review, tissue microarray (TMA) construction and constitution of the trial collection for translational research studies. Review of the cases (n = 754) was performed by a board of expert breast pathologists on a one-week working session with discussion of the difficult cases under a multihead microscope. Tumor characteristics were assessed on whole tissue sections. Immunohistochemical detection of Ki67, EGFR, cytokeratins (CK)5/6 and 14, was performed on TMAs. Results: TNBC were significantly associated with younger age at diagnosis (median age 51yr vs 57.5yr in the ER+/PR- subgroup, p &amp;lt; 0.001). Most tumors were invasive ductal carcinomas (72%). Distribution of special histological subtypes was significantly different in the TNBC and ER+/PR−/HER2− subgroups, with the former comprising medullary (n = 16/17) and metaplastic (n = 34/34) subtypes while the latter was enriched in invasive lobular carcinomas (n = 27/35, p &amp;lt; 0.0001). TNBC were significantly associated with higher histological grade as compared to ER+/PR−/HER2− subgroup (Table 1). Accordingly, TNBC displayed significantly higher proliferative activity as shown by mitotic count and Ki67 index (p &amp;lt; 0.001). As compared to ER+/PR−/HER2− subgroup, TNBC showed distinct characteristics, and displayed a so-called basal-like phenotype in 80%. Among the ER+/PR−/HER2− subgroup, most tumors were classified as luminal B (64%). Interestingly, the presence of tumor lymphocytic infiltrate was more frequently observed in luminal B (59%) as compared to luminal A (30%) subtype (p &amp;lt; 0.001). Conclusion: The Unicancer-PACS08 patient cohort is mainly composed of TNBC that harbour distinct pathological features. Description of the PACS08 collection provides a solid basis for translational research projects, which have been initiated with regards to genomic instability and DNA damage repair, immune system, and biomarker studies to identify new therapeutic targets. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-05-07.