Ratio Of Ki-67 Positive Cells Research Articles

Effects of exosomes from human adipose-derived mesenchymal stem cells on inflammatory response of mouse RAW264.7 cells and wound healing of full-thickness skin defects in mice

Effects of exosomes from human adipose-derived mesenchymal stem cells on inflammatory response of mouse RAW264.7 cells and wound healing of full-thickness skin defects in mice

MP33-09 GUT MICROBIOTA-DERIVED SHORT-CHAIN FATTY ACIDS PROMOTE PROSTATE CANCER GROWTH VIA IGF-1 SIGNALING IN OBESE MICE

MP33-09 GUT MICROBIOTA-DERIVED SHORT-CHAIN FATTY ACIDS PROMOTE PROSTATE CANCER GROWTH VIA IGF-1 SIGNALING IN OBESE MICE

Development of a novel meniscal sheet scaffold and its effectiveness for meniscal regeneration in a rabbit defect model.

This study evaluated the biomechanical strength of a novel two-layer meniscal sheet scaffold (MSS) consisting of polyglycolic acid and poly-Llactic acid/caprolactone and investigated meniscal healing using wrapping treatment for meniscal defect model in a rabbit. The ultimate failure load of the MSS was determined using a tensile testing machine, in vitro. A 2-mm cylindrical defects were created at the medial meniscus of rabbit knees (n = 40). Each knee was assigned to one of two groups. The defect group was not treated and the MSS group underwent wrapping treatment with MSS. Menisci were harvested at 2, 4, 8, and 12 weeks post-implantation. The regenerated meniscus and defect size were evaluated using macrophotographs. Ishida scores for regenerated tissue were determined using Safranin-O/Fast Green staining. Immunohistochemical analysis of Ki-67 for cell proliferation, anti-type I and II collagen antibodies for structure of the regenerated tissue was elucidated. Medial femoral cartilage was stained with Safranin-O/Fast Green and evaluated with Osteoarthritis Research Society International (OARSI) scores. The strength of MSS was maintained over 90% from initial time point to 4 weeks after hydrolysis and over 60% of the strength remained at 8 weeks. The surface area of the meniscus was larger and the defect size smaller in the MSS group than in the defect group at 8 and 12 weeks. Ishida scores revealed that the MSS group improved significantly compared to that of the defect group at all postsurgery time points evaluated. Ki-67 positive cell ratio was significantly higher in the MSS group. OARSI score of the defect group was significantly higher and the defect group showed progressive degeneration in the articular cartilage from 8 to 12 weeks. Overall, wrapping meniscus defects with MSS was useful for accelerating meniscal healing from an early stage and beneficial for tissue regeneration and promoting extracellular matrix maturation.

Inflammatory response or oxidative stress induces upregulation of PTPN2 and thus promotes the progression of laryngocarcinoma.

This study aims to investigate whether inflammatory response or oxidative stress could induce upregulation of PTPN2, thus promoting the progression of laryngocarcinoma. PTPN2 levels in laryngocarcinoma tissues and normal tissues were detected. In addition, PTPN2 levels in laryngocarcinoma tissues with stage 1/2 or stage 3/4 were determined as well. In vitro abundance of PTPN2 was measured in laryngocarcinoma cells and immortalized human nasopharyngeal epithelial cells. Survival analysis was conducted in laryngocarcinoma patients with high or low expression level of PTPN2. Subsequently, M4E cells were stimulated with inflammation (IFN-γ or TNF-α treatment) or oxidative stress (H2O2 stimulation), followed by determination of the protein level of PTPN2. In M4E cells stimulated with different concentrations of H2O2, the clonality and Ki-67 positive cell ratio were detected. Finally, clonality and Ki-67 positive cell ratio in M4E cells transfected with negative control or sh-PTPN2, regardless of H2O2 stimulation, were assessed. PTPN2 was upregulated in laryngocarcinoma tissues, especially those in stage 3/4. Similarly, in vitro abundance of PTPN2 was higher in laryngocarcinoma cell lines. The high level of PTPN2 predicted poor prognosis in laryngocarcinoma patients. IFN-γ or TNF-α treatment upregulated the protein level of PTPN2. Meanwhile, H2O2 stimulation upregulated the protein level of PTPN2, dose-dependently increased clonality, and Ki-67 positive cell ratio in M4E cells. The knockdown of PTPN2 suppressed clonality and Ki-67 positive cell ratio in M4E cells stimulated by H2O2 or not. Inflammatory response or oxidative stress could induce upregulation of PTPN2, thus promoting the proliferative ability of laryngocarcinoma.

Upregulated PTPN2 induced by inflammatory response or oxidative stress stimulates the progression of thyroid cancer

PTPN2 is one of the members of the protein Tyrosine Phosphatases (PTPs) family. To explore the promotive effect of upregulated PTPN2 induced by inflammatory response or oxidative stress on the progression of thyroid cancer. PTPN2 level in thyroid cancer tissues and cell lines was detected. Kaplan-Meier method was applied for evaluating the prognostic value of PTPN2 in thyroid cancer patients. After stimulation of inflammatory response (treatment of IFN-γ and TNF-α), or oxidative stress (treatment of H2O2), protein level of PTPN2 in K1 cells was measured by Western blot. Regulatory effects of PTPN2 on EdU-positive staining and Ki-67 positive cell ratio in K1 cells either with H2O2 stimulation or not were determined. PTPN2 was upregulated in thyroid cancer tissues and cell lines. Its level was higher in metastatic thyroid cancer patients than those of non-metastatic ones. High level of PTPN2 predicted worse prognosis of thyroid cancer. Treatment of either IFN-γ or TNF-α upregulated protein level of PTPN2 in K1 cells. Meanwhile, H2O2 stimulation upregulated PTPN2, which was reversed by NAC administration. With the stimulation of increased doses of H2O2, EdU-positive staining and Ki-67 positive cell ratio were dose-dependently elevated. Silence of PTPN2 attenuated proliferative ability and Ki-67 expression in K1 cells either with H2O2 stimulation or not. Inflammatory response or oxidative stress induces upregulation of PTPN2, thus promoting the progression of thyroid cancer.

Nicotine enhances mesangial cell proliferation and fibronectin production in high glucose milieu via activation of Wnt/β-catenin pathway.

Diabetic nephropathy (DN) is a major complication of diabetes mellitus. Clinic reports indicate cigarette smoking is an independent risk factor for chronic kidney disease including DN; however, the underlying molecular mechanisms are not clear. Recent studies have demonstrated that nicotine, one of the active compounds in cigarette smoke, contributes to the pathogenesis of the cigarette smoking-accelerated chronic kidney disease. One of the characteristics of DN is the expansion of mesangium, a precursor of glomerular sclerosis. In the present study, we examined the involvement of Wnt/β-catenin pathway in nicotine-mediated mesangial cell growth in high glucose milieu. Primary human renal mesangial cells were treated with nicotine in the presence of normal (5 mM) or high glucose (30 mM) followed by evaluation for cell growth. In the presence of normal glucose, nicotine increased both the total cell numbers and Ki-67 positive cell ratio, indicating that nicotine stimulated mesangial cell proliferation. Although high glucose itself also stimulated mesangial cell proliferation, nicotine further enhanced the mitogenic effect of high glucose. Similarly, nicotine increased the expression of Wnts, β-catenin, and fibronectin in normal glucose medium, but further increased mesangial cell expression of these proteins in high glucose milieu. Pharmacological inhibition or genetic knockdown of β-catenin activity or expression with specific inhibitor FH535 or siRNA significantly impaired the nicotine/glucose-stimulated cell proliferation and fibronectin production. We conclude that nicotine may enhance renal mesangial cell proliferation and fibronectin production under high glucose milieus partly through activating Wnt/β-catenin pathway. Our study provides insight into molecular mechanisms involved in DN.

Melatonin mitigates bisphenol A-induced estradiol production and proliferation by porcine ovarian granulosa cells in vitro

Melatonin plays a crucial role in the amelioration of reproductive toxicity induced by endocrine-disrupting chemicals. However, very few studies have investigated the mitigating effects of melatonin on BPA-induced dysfunction in porcine granulosa cells. In the present study, primary granulosa cells were cultured in serum-low conditions with bisphenol A (BPA) (10 μM) with or without melatonin (100 μM), followed by evaluation of estradiol synthesis and cell proliferation. Our results showed that BPA significantly increased estradiol concentration and granulosa cell proliferation. Interestingly, melatonin co-incubation reduced the high levels of estradiol in porcine ovarian granulosa cells induced by BPA stimulation. Furthermore, melatonin co-incubation also attenuated BPA-induced proliferation as shown by a decline in the Ki67-positive cell ratio and PCNA expression level. However, treatment with melatonin-alone did not dramatically reduce estradiol levels or expression of proliferative regulatory protein markers (Ki67, PCNA). We hypothesize that the regulation by melatonin of estradiol biosynthesis and cellular proliferation is highly correlated with BPA stimulation. In conclusion, this study first showed that melatonin mitigated BPA-induced estradiol increase and proliferation in porcine ovarian granulosa cells in vitro. Our results suggest that melatonin may be a promising pharmacologic agent for preventing the potential reproductive toxicity caused by endocrine-disrupting chemicals.

Low-dose DHA-induced astrocyte proliferation can be attenuated by insufficient expression of BLBP in vitro

Docosahexaenoic acid (DHA) is an n-3 long chain polyunsaturated fatty acid (PUFA) that is involved in a wide range of cellular processes in human cells. Brain lipid binding protein (BLBP) exhibits a high affinity for n-3 PUFAs, especially DHA, but the precise functional contributions of DHA and BLBP in astrocytes are not clear. We analyzed cell viability and the ratio of Ki67 positive cells after manipulating DHA and/or BLBP levels in cultured astrocytes, and found that low-dose DHA stimulated proliferation of astrocytes, whereas this proliferative effect could be attenuated by downregulation of BLBP. Moreover, we found that astrocyte proliferation was directly regulated by BLBP independently of DHA. Taken together, low-dose DHA-induced astrocyte proliferation was disturbed by insufficient BLBP; and besides acting as a fatty acid transporter, BLBP was also involved in the proliferation of astrocytes directly.

Sirtuin1 Regulates the Stem Cell Therapeutic Effects on Regenerative Capability for Treating Severe Heart Failure in a Juvenile Animal Model

This study hypothesized that autologous cell transplantation therapy could have greater therapeutic efficacy in juvenile patients than in adult patients, related to differences in sirtuin1 (SIRT1) expression in transplanted cells. A model of heart failure was established in mini-pigs by using coronary artery occlusion. Stem cell cultures were established from juvenile and adult mini-pigs. The cells were prepared as cell sheets and transplanted into corresponding adult or juvenile mini-pigs, and the therapeutic effects were examined invivo and invitro. Next, SIRT1 knockdown cells were established using small interfering RNAs for SIRT1, and the therapeutic effects of the cells were examined invitro and invivo in Lewis rat models of heart failure. Cardiac function showed significantly more improvement in juvenile than in adult pigs. Histologic analysis revealed significant reduction of myocardial fibrosis and hypertrophic response in juvenile pigs. Invitro analysis demonstrated that doubling time was significantly shorter, the ratio of Ki67-positive cells was significantly greater, and the expression of SIRT1, hypoxia-induced factor-1α, hepatocyte growth factor, and stromal cell-derived factor-1 was significantly upregulated in juvenile cells. SIRT1 knockdown cells showed decreased proliferation and cytokine release potential compared with wild-type cells. Transplantation of SIRT1 knockdown stem cell sheets showed lesser improvement of cardiac function aftersevere heart failure in Lewis rats than in controls. Sirtuin1 expression in transplanted cells enhances the skeletal stem cell sheet therapeutic effects for treating severe heart failure in a juvenile animal model.

A new method for meniscus repair using type I collagen scaffold and infrapatellar fat pad.

The aim of this study was to investigate a new method for meniscal repair by combinative transplantation with type I collagen scaffold and infrapatellar fat pad. Two-mm cylindrical defects at the anterior part of bilateral medial menisci were prepared in nine Japanese white rabbits. The 18 knees were equally divided into three groups: I, no treatment; II, collagen scaffold transplantation; and III, collagen scaffold and infrapatellar fat pad transplantation. Another three rabbits (six knees) underwent sham surgery and served as controls. Rabbits were sacrificed at eight weeks after transplantation. Surface area of the medial meniscus was evaluated using macrophotographs. Ishida score for meniscal regeneration was used for assessment. To evaluate the composition of regenerated tissue, immunohistochemistry was analyzed with anti-type I and anti-type II collagen antibodies, and anti-Ki67 antibody. To investigate the effects of collagen scaffold on human meniscus, cells were isolated from human meniscus and infrapatellar fat pad, and cultured with collagen scaffold for three weeks. After that, gene expression was evaluated by using quantitative real-time polymerase chain reaction. In group I, the meniscus shrank anterior to posterior, and the surface area was significantly less than that of normal meniscus. However, the surface area was maintained in group III. Ishida score and Ki67-positive cell ratio in group III were significantly higher than that in any other group, and staining with type I and type II collagen was similar to that of the control. Expression of matrix metalloproteinase was significantly lower in cocultures of collagen scaffold, meniscus cell, and infrapatellar fat pad cell than in monocultured meniscus cell, and expression of interleukin-1β was not increased. This new method for meniscal repair by combinative transplantation with type I collagen scaffold and infrapatellar fat pad showed meniscal regeneration and potential for suppressing inflammation.

Expression of Ki-67, Oct-4, γ-tubulin and α-tubulin in human tooth development

AimsTo analyze factors controlling cell proliferation and differentiation, and appearance of primary cilia during the cap and bell stages of incisor or/and canine human enamel organs. Materials and methodsQualitative and quantitative analysis of proliferating Ki-67 positive cells and expression of γ-tubulin, α-tubulin and Oct-4 was immunohistochemically analyzed in the cap an bell stages of 10 developing human incisor and canine germs, 8–21 weeks old. ResultsDuring the analyzed period, ratio of Ki-67 positive cells changed in outer enamel epithelium from 48.86% to 24.52%, in inner enamel epithelium increased from 56.11% to 60.06% and then dropped to 44.24%. While in dental papilla proliferation first increased from 46.26% to 55.45%, and then dropped to 22.08%, a constant decrease of proliferation characterized enamel reticulum (from 46.26% to 15.49%). Strong cytoplasmic Oct-4 expression characterized epithelial parts of enamel organ, particularly the differentiating ameloblasts. During further development, Oct-4 expression shifted to both nuclear and cytoplasmic expression in mesenchymal tooth components. Primary cilia characterized most of the cells in developing enamel organ. While non-ciliated (proliferating) cells mainly contained two centrioles (γ-tubulin), the primary cilia (α-tubulin) were arising from basal bodies (γ-tubulin) of non-proliferating cells. ConclusionsWe suggest that increase in cell proliferation enables growth of enamel organ, while its selective decrease leads to disintegration of some tooth parts. Drop of proliferation coincided with initiation of ameloblast and odontoblast differentiation. Additionally, cell differentiation was accompanied by increased expression of Oct-4 and probably by signalling via primary cilia, both regulating processes of cell proliferation and differentiation.

Stromal Filters in Automated Immunostain Scoring

KI-67 is a marker for cell proliferation which binds a nuclear antigen and is thus a prime antibody in immunohistochemistry. Scoring methodologies derived from KI-67 immunostaining have shown promise as predictors of lethality in a range of cancers [1]. The most common scoring method is the labelling index, a ratio of KI-67 positive cells to the entire population [1]. Manual calculation of a labelling index can be a time-consuming process for pathologists, who count cells in a bright field. Automated scoring algorithms and programs have been written to generate labelling indices, but they are nonspecific with respect to cell type and most often skew results by including stromal cells in the index. This problem is particularly relevant in breast cancer, where tumors are often located in a field of fibroadipose tissue. We have adapted algorithms for immunostain scoring and tested 2 stromal cell filtration algorithms which remove these cells based on their elongated nuclear morphology.

Abstract 2695: Olfactomedin 4 gene is associated with progression of prostate cancer: Evidence from an OLFM4 knockout mouse model

Abstract The olfactomedin 4 gene (OLFM4) encodes an olfactomedin-related glycoprotein whose physiological and pathological functions are largely unknown. We have previously reported that expression of OLFM4 is down regulated during the progression of human prostate cancer, and that reduced expression is due to gene deletions of OLFM4 (36.7%) and/or hyper-methlytion of CpG sites in the promoter region. We also demonstrated that ectopic expression of OLFM4 in several human prostate cancer cell lines inhibits proliferation, invasion and metastatic potential. In this study, we further investigated the potential role of OLFM4 in the carcinogenesis of prostate cancer utilizing an OLFM4 knock out mouse model. Histopathological analysis revealed that homozygous OLFM4 mutant mice develop prostatic epithelial lesions in an age-dependent manner. The prostatic epithelial hyperplasia was seen at 6 months of age, prostatic intraepithelial neoplasia (PIN) was found at 10-12 months of age, and prostate adenoma was observed from 18-24 months of ages in the both anterior prostate (AP) and dorsal-lateral prostate (DLP) of OLFM4-null mice. The frequency of PIN lesions at the AP and DLP was 0 % (0/20) at 3-6 months, 38% (5/13) at 10-12 months and 44% (11/25) at 18-24 months. The frequency of prostatic adenoma was 28% (7/25), and 8% (2/25) of OLFM4-null mice developed lung metastases at 18-24 months of age. To determine whether OLFM4 affects proliferation or apoptosis of prostatic epithelial cells in the OLFM4-null mice, we carried out Ki-67 immunohistochemical staining and TUNEL assays. The ratio of Ki-67 positive cells was significantly higher at 3-6 months (p=0.04180), 10-12 months (P=0.002) and 18-20 months (p=0.0038) compared to age-matched controls, OLFM4 wild type mice, or heterozygous mice, thus indicating an increase in proliferation in OLFM4-null prostate tissues. There was no difference in the number of apoptotic cells in the control versus null mice. Since we have previously found that OLFM4 blocks CXCL12 induced AKT activity in the human prostate cancer cell PC-3, and inhibits cell invasion, we studied the phosphorylation of AKT and downstream signaling pathway components by Western blot analysis of prostate samples from 3-4 month old mice. Compared with OLFM4 wild type mice, pAKT, pGSK3β, β-catenin, cyclin D1 and c-Myc were increased in the OLFM4-null mice tissue even in the absence of morphological abnormalities. Elevated β-catenin protein in OLFM4-null mice prostate epithelial cells was also observed in PIN and tumor cells at 18 months of age, and positive staining with Cyclin D1 in OLFM4-null tumor was also detected. Taken together, the data show OLFM4-null prostatic epithelial cells exhibit higher levels of AKT and β-catenin, suggesting this is the mechanism leading to their enhanced cellular proliferation and carcinogenetic potential. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2695. doi:1538-7445.AM2012-2695

Immunohistochemical Assessment of Ki-67 Expression in Adenoid Cystic Carcinoma of the Salivary Glands

Objective Adenoid cystic carcinoma (ACC) is a rare malignant tumor originating from the salivary glands. It’s rather bland histological appearance that masks its ultimate biological aggressiveness. Evaluation of cell cycle and mitoses have been useful in predicting malignancy in many tumors. Ki-67 antigen is a human nuclear antigen that presents during all active phases of cell cycle. The aim of this study was to estimate whether the Ki-67 expression ratio in ACC correlated with the morphological growth pattern and tumor histological grade. Material and Methods Tissue samples of 19 ACC, collected from the files in archive of Department of oral pathology, Mashhad Dentistry Faculty. All Samples originated from minor salivary gland including 11 men and 8 women with an avarage age of 46. One section have been stained with H&E to confirm the diagnosis and the other with Ki - 67 monoclonal antibody. All samples graded and scored for Ki-67 immunoreactivity, then the ratio of Ki-67 positive cells was calculated. Results The most incidence of tumor was in 4 and 5 decades and in women. The most common site of tumors was palate. Ki-67 expressed in 68% of all samples. The Ki-67 immunoreativity ranges from 15% to 85%. Although the avarage percentage of Ki-67 expression seems to increase with histological grade, but the difference between grade III and grade I, and between grade III and mixed I / II was not statistically significant (P value = 0.3). Conclusion For ACC, Ki-67 immunostaining regarding to histological grading is not a reliable tool in predicting the intensity of tumor aggressiveness and seems to have less value. Further studies with greater series of samples are needed to confirm this issue.