The effects of acute and chronic administration of phosphatidylserine on cell proliferation and survival in the dentate gyms of adult and middle-aged rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Immunoexpression of angiogenesis and proliferation markers in patients treated with cyclosporin A

In the present immunohistochemical study, the expression of vascular endothelial growth factor, nitric oxide synthase 1 and 3, and Ki-67 in the gingival tissues of renal transplant patients treated with cyclosporin A was assessed. Gingival overgrowth (GO) frequently occurs in transplant patients receiving immunosuppressive drugs such as cyclosporine and this gingival inflammation might play an important role in the pathogenesis of drug-induced GO. Twenty-eight human gingival biopsies were taken from healthy patients with chronic periodontitis (N.=14 control group), and from renal transplant recipients treated with cyclosporin A (N.=14 test group). The retrieved specimens were immunohistochemically processed and stained for vascular endothelial growth factor, nitric oxide synthase 1 and 3, and Ki-67. The levels of vascular endothelial growth factor, nitric oxide synthase 1 and 3, and Ki-67 were found to be significantly different among groups (P>0.001), with patients treated with cyclosporin A showing higher levels of all the analyzed markers compared to control group. In summary, the data from this pilot study suggests that the investigated factors have a role in the inflammation processes associated to immunosuppressive therapy. However, further studies with a larger sample population need to be conducted for an exhaustive knowledge of the mechanisms leading to GO.

Clopidogrel enhances mesenchymal stem cell proliferation following periodontitis

Bone formation is dependent on the differentiation of osteoblasts from mesenchymal stem cells (MSCs). In addition to serving as progenitors, MSCs reduce inflammation and produce factors that stimulate tissue formation. Upon injury, MSCs migrate to the periodontium, where they contribute to regeneration. We examined the effect of clopidogrel and aspirin on MSCs following induction of periodontitis in rats by placement of ligatures. We showed that after the removal of ligatures, which induces resolution of periodontal inflammation, clopidogrel had a significant effect on reducing the inflammatory infiltrate. It also increased the number of osteoblasts and MSCs. Mechanistically, the latter was linked to increased proliferation of MSCs in vivo and in vitro. When given prior to inducing periodontitis, clopidogrel had little effect on MSC or osteoblasts numbers. Applying aspirin before or after induction of periodontitis did not have a significant effect on the parameters measured. These results suggest that clopidogrel may have a positive effect on MSCs in conditions where a reparative process has been initiated.

Aedes aegypti proliferation in stormwater retained in Best Management Practices

The present study aimed to evaluate the risk of Aedes aegypti proliferation in structures used in compensatory techniques for urban drainage (Best Management PracticesBMPs). These drainage structures are utilised to reduce flood peaks due to surface runoff, and they have been used in many countries. However, many of these structures have been designed to keep water surfaces exposed for a certain period of time, depending on the type of project. Exposed water surfaces may become an ideal environment for A. aegypti proliferation in tropical and subtropical areas where the rainy season occurs during the summer. Thus, data regarding the mosquito life cycle, consecutive rainfall pattern and emptying time of these structures were collected. A comparison of these data led to the evaluation of the associated risk of A. aegypti proliferation in BMP structures. The risk of mosquito proliferation ranged from 1.1% to 3.3%, depending on the rainfall pattern, A. aegypti life cycle phase and BMP activity.

Correlation between immunoexpression of BCL-2 protein family with apoptosis index, cellular proliferation and survival in colorectal carcinomas

Objectives: To evaluate the Bcl-2, Bax, Bad and Bak immunoexpression in tumor and nontumorous tissue of 130 patients with colorectal carcinoma submitted to surgery at São Paulo Hospital, EPM/ UNIFESP, from 2002 to 2005, and to correlate the immunoexpression data with the apoptotic index (AI, obtained by anti-cleaved caspase 3 and M30 labeling), cell proliferation score (CPS, obtained by Ki-67), immunoexpression of p53 and patient’s clinical prognosis. Results: Positive correlation was verified between Bcl-2 protein family in tumor and nontumor tissue. Only Bcl-2 protein correlated with IA and CPS in the tumor. Positive correlation was observed between pro- -apoptotic proteins and Bcl-2 protein. In the adjacent mucosa, Bcl-2 correlated with Ki-67 and p53, but not with IA. Carcinomas exhibited higher immunoexpression of CPS and IA markers. No correlation occurred between immunoexpression data and patient survival. Conclusion: Positive correlation was observed between the pro-apoptotic proteins of the Bcl-2 family and the anti-apoptotic protein Bcl-2. In the adjacent nontumor mucosa, Bcl-2 correlated with Ki-67 and p53, but not with AI. Carcinomas presented greater immunoexpression for CPS and AI markers; however immunoexpression of these markers was not correlated with patient survival.

Nitric oxide is responsible for oxidative skin injury and modulation of cell proliferation after 24 hours of UVB exposures

Nitric oxide (NO) is produced by various mammalian cells and plays a variety of regulatory roles in normal physiology and in pathological processes. This article provides evidence regarding the participation of NO in UVB-induced skin lesions and in the modulation of skin cell proliferation following UVB skin irradiation. Hairless mice were subjected to UVB irradiation for 3 hours and the skin evaluated immediately, 6 and 24 hours postirradiation. The skin lipid peroxidation, and NO levels evaluated by chemiluminescence and inducible nitric oxide synthase (iNOS) and nitrotyrosine immunolabelling increased significantly 24 hours after irradiation and decreased under the treatment with aminoguanidine (AG). On the other hand, cell proliferation markers, PCNA and VEGF showed a strong labelling index when AG was used. The data indicate that NO mediates, at least in part, the lipid peroxidation and protein nitration and also promotes the down regulation of factors involved in cell proliferation. This work shows that the NO plays an important role in the oxidative stress damage and on modulation of cell proliferation pathways in UVB irradiated skin.

Fibronectin glycation increases IGF-I induced proliferation of human aortic smooth muscle cells

The advanced glycation end products, namely AGEs, contribute to long-termed complications of diabetes mellitus, including macroangiopathy, where smooth muscle cells (SMC) proliferation stimulated by platelet-derived growth factor (PDGF) isoforms and insulin-like growth factor-I (IGF-I) plays an important role. The objective of the present study was to investigate the effect of an AGE-modified extracellular matrix protein on IGF-I induced SMC proliferation and on the IGF-I-IGF binding protein 4 (IGFBP-4) axis under basal conditions and after stimulation with PDGF-BB. IGF-I resulted in significantly higher thymidine incorporation in SMC seeded on AGE-modified fibronectin (AGE-FN) in comparison to cells seeded on fibronectin (FN). This augmented proliferation could not be accounted for by increased expression of IGF-IR, by decreased secretion of IGFBP-4, a binding protein that inhibits IGF-I mitogenic effects or by increased IGF-IR autophosphorylation. PDGF-BB did not modulate IGF-IR and IGFBP-4 mRNA expression in any of the substrata, however, this growth factor elicited opposite effects on the IGFBP-4 content in the conditioned media, increasing it in cells plated on FN and diminishing it in cells plated on AGE-FN. These findings suggest that one mechanism by which AGE-modified proteins is involved in the pathogenesis of diabetes-associated atherosclerosis might be by increasing SMC susceptibility to IGF-I mitogenic effects.

Effect of low-level laser therapy after rapid maxillary expansion on proliferation and differentiation of osteoblastic cells

The aim of this study was to investigate the osteoblastic activity of cells derived from the midpalatal suture upon treatment with low-level laser therapy (LLLT) after rapid maxillary expansion (RME). A total of 30 rats were divided into two groups: experimental I (15 rats with RME without LLLT) and experimental II (15 rats with RME + LLLT). The rats were euthanized at 24 h, 48 h, and 7 days after RME, when the osteoblastic cells derived from the rats' midpalatal suture were explanted. These cells were cultured for periods up to 17 days, and then in vitro osteogenesis parameters and gene expression markers were evaluated. The cellular doubling time in the proliferative stage (3-7 days) was decreased in cultured cells harvested from the midpalatal suture at 24 and 48 h after RME + LLLT, as indicated by the increased growth of the cells in a culture. Alkaline phosphatase activity at days 7 and 14 of the culture was increased by LLLT in cells explanted from the midpalatal suture at 24 and 48 h and 7 days after RME. The mineralization at day 17 was increased by LLLT after RME in all periods. Results from the real-time PCR demonstrated that cells harvested from the LLLT after RME group showed higher levels of ALP, Runx2, osteocalcin, type I collagen, and bone sialoprotein mRNA than control cells. More pronounced effects on ALP activity, mineralization, and gene expression of bone markers were observed at 48 h after RME and LLLT. These results indicate that the LLLT applied after RME is able to increase the proliferation and the expression of an osteoblastic phenotype in cells derived from the midpalatal suture.

Protein restriction inhibits gastric cell proliferation during rat postnatal growth in parallel to ghrelin changes

Objective: Gastric development depends directly on the proliferation and differentiation of epithelial cells, and these processes are controlled by multiple elements, such as diet, hormones, and growth factors. Protein restriction affects gastrointestinal functions, but its effects on gastric growth are not fully understood. Methods: The present study evaluated cell proliferation in the gastric epithelia of rats subjected to protein restriction since gestation. Because ghrelin is increasingly expressed from the fetal to the weaning stages and might be part of growth regulation, its distribution in the stomach of rats was investigated at 14, 30, and 50 d old. Results: Although the protein restriction at 8% increased the intake of food and body weight, the body mass was lower (P < 0.05). The stomach and intestine were also smaller but increased proportionately throughout treatment. Cell proliferation was estimated through DNA synthesis and metaphase indices, and lower rates (P < 0.05) were detected at the different ages. The inhibition was concomitant with a larger number of ghrelin-immunolabeled cells at 30 and 50 d postnatally. Conclusion: Protein restriction impairs cell proliferation in the gastric epithelium, and a ghrelin upsurge under this condition is parallel to lower gastric and body growth rates. (C) 2012 Elsevier Inc. All rights reserved.

Ursolic acid directly promotes protein accretion in myotubes but does not affect myoblast proliferation

Ursolic acid (UA) has been recently proposed as a potential candidate for the treatment of muscle wasting conditions because of its protein sparring/anabolic effects. Despite this finding, it is unknown whether this response is the consequence of a direct effect on the muscle fibre or if it is mediated by neural or other systemic factors. In the present study, we sought to determine if UA has direct effects in skeletal muscle cells, whether it can increase myoblast proliferation and whether UA can become myotoxic at higher doses. Our results demonstrate that UA directly promoted protein accretion in cultured myotubes but did not modulate myoblast proliferation. At higher doses, UA compromised cell viability in both myoblasts and myotubes. We conclude that the anabolic properties of UA seen in vivo and in vitro are likely a direct effect on the muscle cell, but at higher doses, the benefits decline in favour of a myotoxic outcome. Copyright (C) 2012 John Wiley & Sons, Ltd.

In Vitro PLK1 Inhibition by BI 2536 Decreases Proliferation and Induces Cell-Cycle Arrest in Melanoma Cells

Melanoma is one of the most treatment-resistant malignancies and regardless of new therapeutic tactics the outcome remains dismal. Polo-like kinase 1 (PLK1) has been shown to be over-expressed in a variety of tumors, becoming an attractive target for cancer management. In the present study we tested the in vitro antitumor activities of BI 2536, a selective inhibitor of PLK1, against two melanoma cell lines. Our results showed that nanomolar concentrations (10-150 nmol/L) of the drug significantly decreased cell proliferation and clonogenicity, promoting cell cycle arrest in G2/M. Targeting the cell cycle offers an attractive potential cancer-treatment option. Herein we show that PLK1 inhibition may be a feasible approach for the impairment of tumor progression and dissemination. This in vitro profile of melanoma cell growth inhibition by PLK1 modulation may be an interesting model to be tested in association with first-line antineoplasic agents in melanomas.

High-Fat Diet Obesity Associated With Insulin Resistance Increases Cell Proliferation, Estrogen Receptor, and PI3K Proteins in Rat Ventral Prostate

In this study, we evaluated the effects of obesity and insulin resistance induced by a high-fat diet on prostate morphophysiology, focusing on cell proliferation, expression of androgen (AR) and estrogen receptors (ER) and proteins of the insulin signaling pathway. Adult male Wistar rats were fed a high-fat diet (20% fat) for 15 weeks, whereas control animals received a balanced diet (4% fat). Both groups were then divided and treated for 2 weeks with 1 mg/kg body weight/day of the aromatase inhibitor letrozole or vehicle only. The ventral prostate was analyzed with immunohistochemical, histopathological, stereological, and Western blotting methods. Obese rats showed insulin resistance, hyperinsulinemia, and reduced plasma testosterone levels. The incidence of prostatic intraepithelial neoplasia (PIN) was 2.7 times higher in obese rats and affected 0.4% of the gland compared with 0.1% PIN areas found in control rats. Obesity doubled cell proliferation in both prostate epithelium and stroma. AR content decreased in the prostate of obese rats and estrogen receptor beta (ER beta) increased in this group. Protein levels of insulin receptor substrate 1 and protein kinase B diminished in the obese group, whereas phosphatidylinositol 3-kinase (PI3K) increased significantly. Most structural changes observed in the prostate of obese rats normalized after letrozole treatment, except for increased stromal cell proliferation and ER beta expression, which might be associated with insulin resistance. This experimental model of obesity and insulin resistance induced by a high-fat diet increases cell proliferation in rat prostate. Such alterations are associated with decreased levels of AR and increased ER beta and PI3K proteins. This change can facilitate the establishment of proliferative lesions in rat prostate.

Cancer cell proliferation is inhibited by specific modulation frequencies

BACKGROUND: There is clinical evidence that very low and safe levels of amplitude-modulated electromagnetic fields administered via an intrabuccal spoon-shaped probe may elicit therapeutic responses in patients with cancer. However, there is no known mechanism explaining the anti-proliferative effect of very low intensity electromagnetic fields. METHODS: To understand the mechanism of this novel approach, hepatocellular carcinoma (HCC) cells were exposed to 27.12 MHz radiofrequency electromagnetic fields using in vitro exposure systems designed to replicate in vivo conditions. Cancer cells were exposed to tumour-specific modulation frequencies, previously identified by biofeedback methods in patients with a diagnosis of cancer. Control modulation frequencies consisted of randomly chosen modulation frequencies within the same 100 Hz-21 kHz range as cancer-specific frequencies. RESULTS: The growth of HCC and breast cancer cells was significantly decreased by HCC-specific and breast cancer-specific modulation frequencies, respectively. However, the same frequencies did not affect proliferation of nonmalignant hepatocytes or breast epithelial cells. Inhibition of HCC cell proliferation was associated with downregulation of XCL2 and PLP2. Furthermore, HCC-specific modulation frequencies disrupted the mitotic spindle. CONCLUSION: These findings uncover a novel mechanism controlling the growth of cancer cells at specific modulation frequencies without affecting normal tissues, which may have broad implications in oncology. British Journal of Cancer (2012) 106, 307-313. doi:10.1038/bjc.2011.523 www.bjcancer.com Published online 1 December 2011 (C) 2012 Cancer Research UK

NADPH Phagocyte Oxidase Knockout Mice Control Trypanosoma cruzi Proliferation, but Develop Circulatory Collapse and Succumb to Infection

(NO)-N-center dot is considered to be a key macrophage-derived cytotoxic effector during Trypanosoma cruzi infection. On the other hand, the microbicidal properties of reactive oxygen species (ROS) are well recognized, but little importance has been attributed to them during in vivo infection with T. cruzi. In order to investigate the role of ROS in T. cruzi infection, mice deficient in NADPH phagocyte oxidase (gp91(phox-/-) or phox KO) were infected with Y strain of T. cruzi and the course of infection was followed. phox KO mice had similar parasitemia, similar tissue parasitism and similar levels of IFN-gamma and TNF in serum and spleen cell culture supernatants, when compared to wild-type controls. However, all phox KO mice succumbed to infection between day 15 and 21 after inoculation with the parasite, while 60% of wild-type mice were alive 50 days after infection. Further investigation demonstrated increased serum levels of nitrite and nitrate (NOx) at day 15 of infection in phox KO animals, associated with a drop in blood pressure. Treatment with a NOS2 inhibitor corrected the blood pressure, implicating NOS2 in this phenomenon. We postulate that superoxide reacts with (NO)-N-center dot in vivo, preventing blood pressure drops in wild type mice. Hence, whilst superoxide from phagocytes did not play a critical role in parasite control in the phox KO animals, its production would have an important protective effect against blood pressure decline during infection with T. cruzi.

Regulatory T Cells Accumulate in the Lung Allergic Inflammation and Efficiently Suppress T-Cell Proliferation but Not Th2 Cytokine Production

Foxp3(+)CD25(+)CD4(+) regulatory T cells are vital for peripheral tolerance and control of tissue inflammation. In this study, we characterized the phenotype and monitored the migration and activity of regulatory T cells present in the airways of allergic or tolerant mice after allergen challenge. To induce lung allergic inflammation, mice were sensitized twice with ovalbumin/aluminum hydroxide gel and challenged twice with intranasal ovalbumin. Tolerance was induced by oral administration of ovalbumin for 5 consecutive days prior to OVA sensitization and challenge. We detected regulatory T cells (Foxp3(+)CD25(+)CD4(+) T cells) in the airways of allergic and tolerant mice; however, the number of regulatory T cells was more than 40-fold higher in allergic mice than in tolerant mice. Lung regulatory T cells expressed an effector/memory phenotype (CCR4(high)CD62L(low)CD44(high)CD54(high)CD69(+)) that distinguished them from naive regulatory T cells (CCR4(int)CD62L(high)CD44(int)CD54(int)CD69(-)). These regulatory T cells efficiently suppressed pulmonary T-cell proliferation but not Th2 cytokine production.