Cedrus atlantica Extract Suppress Glioblastoma Growth through Promotion of Genotoxicity and Apoptosis: In Vitro and In Vivo Studies

Glioblastoma (GBM) is the most common malignant primary brain tumor in humans, exhibiting highly infiltrative growth and drug resistance to conventional chemotherapy. Cedrus atlantica (CAt) extract has been shown to decrease postoperative pain and inhibit the growth of K562 leukemia cells. The aim of this study was to assess the anti-GBM activity and molecular mechanism of CAt extract in vitro and in vivo. The results showed that CAt extract greatly suppressed GBM cells both in vitro and in vivo and enhanced the survival rate in subcutaneous and orthotopic animal models. Moreover, CAt extract increased the level of ROS and induced DNA damage, resulting in cell cycle arrest at the G₀/G₁ phase and cell apoptosis. Western blotting results indicated that CAt extract regulates p53/p21 and CDK4/cyclin D1 protein expression and activates extrinsic and intrinsic apoptosis. Furthermore, CAt extract enhanced the cytotoxicity of Temozolomide and decreased AKT/mTOR signaling by combination treatment. In toxicity assays, CAt extract exhibited low cytotoxicity toward normal cells or organs in vitro and in vivo. CAt extract suppresses the growth of GBM by induction of genotoxicity and activation of apoptosis. The results of this study suggest that CAt extract can be developed as a therapeutic agent or adjuvant for GBM treatment in the future.     

Anti-Inflammatory Effect of Lemon Mucilage: In Vivo and In Vitro Studies

The mucilage extracted from a lemon juice centrifugation pulp was studied for its anti-inflammatory effect in rat. In vivo the lemon mucilage significantly inhibited carrageenan-induced edema in rat paw from 59% to 73.5% showing the highest effect at the third hour. In vitro, at the doses of 10^?8, 10^?6, 10^?4 or 10^?2 mg/mL the lemon mucilage stimulated the superoxide anion production in rat testing neutrophils in whole blood but inhibited it in FMLP stimulated cells at the dose of 10^?2 mg/mL. The neutrophils of rats receiving p.o. the lemon mucilage for 21 days showed a significant decrease of 45.5% in O₂^? generation after FMLP stimulation, and a not-significant increase after phorbol-12-myristate-13-acetate (PMA) or zymosan stimulation. Since the activity on zymosan- and PMA-induced O₂^? production was not significant, the inhibition exerted by FMLP in rat neutrophils occurred mainly through the blockade of phospholipase D.     

Corticosteroids, Serum, and Phagocytosis: In Vitro and In Vivo Studies

The phagocytic-bactericidal capacity (PBC) of human polymorphonuclear leukocytes (PMNs) for strains of Klebsiella (K) and of Enterococcus (E) was unaffected in vitro by the presence of 100 mug of either hydrocortisone (HC) or of methylprednisolone (MP) per ml in the medium. At higher concentrations (500 to 2,000 mug/ml) both compounds impaired PBC-K and PBC-E, but the latter was less sensitive to steroid-induced inhibition. In addition to interfering with intracellular killing of both organisms by PMNs, 2,000 mug of HC per ml also inhibited ingestion of E, although not of K. Steroid-induced inhibition of PBC-K in vitro was completely abolished by increasing the concentration of serum used as opsonin. The PBC-K of human PMNs obtained 30 min after intravenous injection of 1 g of MP was unimpaired in vitro in the presence of 10 to 90% simultaneously obtained autologous serum containing 42 mug of MP per ml. These findings suggest that short-term, high-dosage administration of MP is unlikely to produce clinically significant impairment of intraleukocytic bacterial killing.     

In Vitro and In Vivo Effects of Endotoxin on Mouse Peritoneal Cells

The in vitro effect of endotoxin (LPS) on unfractionated mouse peritoneal cells and cells fractionated into glass adherent and nonadherent populations was studied. LPS caused blast transformation and deoxyribonucleic acid synthesis in the nonadherent, nonphagocytic cells. There was no evidence of a mitogenic effect on macrophages. Instead, a cytotoxic effect was noted. When incubated with unfractionated peritoneal cells, LPS was still cytotoxic for macrophages, but the mitogenic effect for nonadherent cells was decreased or ablated. The intraperitoneal administration of LPS to mice resulted in an acute inflammatory response with a transient depletion of mononuclear cells. There was no stimulation of division of macrophages. Data were obtained which indicated that local cell division is an important factor in the normal turnover of peritoneal macrophages.     

In Vitro and In Vivo Cardiomyogenic Differentiation of Amniotic Fluid Stem Cells

Cell therapy has developed as a complementary treatment for myocardial regeneration. While both autologous and allogeneic uses have been advocated, the ideal candidate has not been identified yet. Amniotic fluid-derived stem (AFS) cells are potentially a promising resource for cell therapy and tissue engineering of myocardial injuries. However, no information is available regarding their use in an allogeneic context. c-kit-sorted, GFP-positive rat AFS (GFP-rAFS) cells and neonatal rat cardiomyocytes (rCMs) were characterized by cytocentrifugation and flow cytometry for the expression of mesenchymal, embryonic and cell lineage-specific antigens. The activation of the myocardial gene program in GFP-rAFS cells was induced by co-culture with rCMs. The stem cell differentiation was evaluated using immunofluorescence, RT-PCR and single cell electrophysiology. The in vivo potential of Endorem-labeled GFP-rAFS cells for myocardial repair was studied by transplantation in the heart of animals with ischemia/reperfusion injury (I/R), monitored by magnetic resonance imaging (MRI). Three weeks after injection a small number of GFP-rAFS cells acquired an endothelial or smooth muscle phenotype and to a lesser extent CMs. Despite the low GFP-rAFS cells count in the heart, there was still an improvement of ejection fraction as measured by MRI. rAFS cells have the in vitro propensity to acquire a cardiomyogenic phenotype and to preserve cardiac function, even if their potential may be limited by poor survival in an allogeneic setting.     

阳极氧化活化处理纯钛经皮种植体的体内外实验研究

为解决经皮器械在负重条件下的长期稳定和经皮密封问题，本文对比研究了阳极氧化表面以及光滑表面的纯钛种植体经皮植入动物体内的情况．并同时将人皮肤表皮细胞接种于这两种表面上进行培养，初步探讨了阳极氧化活化处理后的纯钛金属作为经皮植入体的可能性。结果表明：经皮部分钛金属的光滑表面与阳极氧化的微观粗糙表面对于术后炎性反应的影响无明显差异，阳极氧化活化表面不仅能与骨形成牢固结合，而且也可以与皮下组织紧密贴附。同时，在体内种植体表面形成的钙磷层可能也是形成经皮密封的原因之一，阳极氧化活化钛的微孔粗糙表面对表皮也有一定的锁合固定作用。因此阳极氧化的表面活化方法有可能成为有效的解决经皮生物密封的活化方法之一。     

Menthol in Combination with Iontophoresis Promotes Natamycin Penetration through the Cornea: In Vitro and In Vivo Studies

Bulletin of Experimental Biology and Medicine - We studied whether menthol can promote penetration of natamycin, a representative antifungal macrolide agent, through the cornea. Natamycin...     

Increased SSeCKS Expression in Rat Hepatic Stellate Cells Upon Activation In Vitro and In Vivo

Recent reports suggest that src suppressed c kinase substrates (SSeCKS) are early inflammatory response protein. However, there is only scarce knowledge on the functional role of SSeCKS in liver under conditions of acute inflammation. In the present study, we investigated SSeCKS expression in liver after administration of carbon tetrachloride (CCl₄) in rats and in isolated primary hepatic stellate cells (HSCs) upon activation on a plastic dish. We found that SSeCKS mRNA was hardly detectable in healthy liver tissue and further increased in carbon tetrachloride-mediated acute liver failure. SSeCKS protein expression was mainly found in hepatic stellate cells. In vitro, SSeCKS expression in activated rat HSCs was dramatically increased. The upregulation of SSeCKS protein expression in rat HSCs during activation in vitro and in vivo suggested the possibility of SSeCKS, an important part of function of the activated HSCs, perhaps through modulation of liver regeneration or formation of liver fibrosis after various injuries.     

In Vivo and In Vitro Studies of Delayed-Type Hypersensitivity to Toxoplasma gondii in Guinea Pigs

Delayed-type hypersensitivity develops late in the course of human toxoplasmosis, and a positive skin test is of some value for implicating chronic or eliminating acute forms of toxoplasmosis as a cause of disease. Toxoplasma-infected guinea pigs were studied to determine the onset and development of delayed-type hypersensitivity. Both the toxoplasmin skin test and the in vitro macrophage migration inhibition technique indicated that delayed hypersensitivity to toxoplasma antigen existed as early as 1 week after infection. The mechanism responsible for the observed inhibition of macrophage migration in vitro appeared to be an inhibitory factor(s) released from sensitized lymphoid cells in the presence of antigen.     

Transdermal Delivery of Amino Acids and Antioxidants Enhance Collagen Synthesis: In Vivo and In Vitro Studies

One of the most visible changes associated with the aging process in humans relates to a progressive thinning of the skin. This results from a decline in both collagen and glycosaminoglycans, as well as from changes in their chemical structure and 3-dimentional organization. Transdermal administration of antioxidants, α -lipoic acid (LA) (0.5%) and proanthocyanidin PA) (0.3%) in a standard cosmetic vehicle base formulation supplemented with 2% benzyl alcohol as a penetration enhancer, a mixture of essential amino acids (0.2%), significantly enhanced collagen synthesis and deposition. The amino acid mixture was designed to mimic serum concentrations, with supplemental methionine added to provide additional sulfur. The histological appearance of the skin of mature female rats treated in this fashion reflected the increased deposition of collagen in the dermis as well as a thickened epidermal layer. The changes do not seem to be mediated by TGF- β or PDGF, two growth factors known to stimulate collagen synthesis. At lower concentrations, α -lipoic acid did not affect cell proliferation but at higher doses, while it had an inhibitory effect on ³H-thimidine uptake, it did enhance collagen production. Pronanthocyanidin did not affect cell proliferation but significantly increased collagen synthesis by cultured fibroblasts.     

Differentiation of Human Embryonic Stem Cells to Cardiomyocytes for In Vitro and In Vivo Applications

The ability of human embryonic stem cells to differentiate into spontaneously contracting cardiomyocyte-like cells has attracted substantial interest from the scientific community over the last decade. From having been difficult to control, human cardiomyogenesis in vitro is now becoming a process which, to a certain extent, can be effectively manipulated and directed. Although much research remains, new and improved protocols for guiding pluripotent stem cells to the cardiomyocyte lineage are accumulating in the scientific literature. However, the stem cell derived cardiomyocytes described to date, generally resemble immature embryonic/fetal cardiomyocytes, and they are in some functional and structural aspects different from adult cardiomyocytes. Thus, a future challenge will be to design strategies that eventually may allow the cells to reach a higher degree of maturation in vitro. Nevertheless, the cells which can be prepared using current protocols still have wide spread utility, and they have begun to find their way into the drug discovery platforms used in the pharmaceutical industry. In addition, stem cell derived cardiomyocytes and cardiac progenitors are anticipated to have a tremendous impact on how heart disease will be treated in the future. Here, we will discuss recent strategies for the generation of cardiomyocytes from human embryonic stem cells and recapitulate their features, as well as highlight some in vitro applications for the cells. Finally, opportunities in the area of cardiac regenerative medicine will be illustrated.     

Anthelmintic Efficacy of Nauclea Latifolia Extract Against Gastrointestinal Nematodes of Sheep: In Vitro and In Vivo Studies

Direct effects of Nauclea latifolia extracts on different gastrointestinal nematodes of sheep is described. In vivo and in vitro studies were conducted to determine possible anthelmintic effect of leaf extracts of Nauclea latifolia toward different ovine gastro intestinal nematodes. A larval development assay was used to investigate in vitro, the effect of aqueous and ethanolic extracts of N. latifolia towards strongyles larvae. The development and survival of infective larvae (L₃) was assessed and best-fit LC₅₀ values were computed by global model of non-linear regression analysis curve-fitting (95% CI). Twenty sheep harbouring naturally acquired gastrointestinal nematodes were treated with oral administration of ethanolic extracts at a dose rate of 125 mg/kg, 250 mg/kg and 500mg/kg to evaluate therapeutic efficacy, in vivo.The presence of the extracts in the cultures decreased the survival of larvae. The LC₅₀ of aqueous and ethanolic extract were 0.704 and 0.650 mg/ml respectively and differ significantly (P<0.05, paired t test). Faecal egg counts (FEC) on day 12 after treatment showed that the extract is effective, relative to control (1-way ANOVA, Dunnett''s multiple comparison test), at 500mg/kg against Haemonchus spp, Trichostrongylus spp (p<0.05), Strongyloides spp (P < 0.01); at 250mg/kg against Trichuris spp (P < 0.01) and ineffective against Oesophagostomum spp (p>0.05). The effect of doses is extremely significant; the day after treatment is sometimes significant while interaction between dose and day after treatment is insignificant (2-way ANOVA).N. latifolia extract could therefore find application in the control of helminth in livestock, by the ethnoveterinary medicine approach.     

Anti-septic Effects of Fisetin In Vitro and In Vivo

Sepsis is a state of disrupted inflammatory homeostasis that is initiated by infection. High mobility group box 1 (HMGB1) protein acting as a late mediator of severe vascular inflammatory conditions, such as sepsis and endothelial cell protein C receptor (EPCR), is involved in vascular inflammation. Fisetin, an active compound from the family Fabaceae, was reported to have antiviral, neuroprotective, and anti-inflammatory activities. Here, we determined the anti-septic effects of fisetin on HMGB1-mediated inflammatory responses and on the shedding of EPCR in vitro and in vivo, for the first time. First, we monitored the effects of post-treatment fisetin on lipopolysaccharide (LPS) and cecal ligation and puncture (CLP)-mediated release of HMGB1 and HMGB1-mediated regulation of pro-inflammatory responses in human umbilical vein endothelial cells (HUVECs) and septic mice. Post-treatment fisetin was found to suppress LPS-mediated release of HMGB1 and HMGB1-mediated cytoskeletal rearrangements. Fisetin also inhibited HMGB1-mediated hyperpermeability and leukocyte migration in septic mice. Fisetin induced potent inhibition of phorbol-12-myristate 13-acetate (PMA) and CLP-induced EPCR. Fisetin also inhibited the expression and activity of tumor necrosis factor-α converting enzyme, induced by PMA in endothelial cells. In addition, fisetin inhibited the production of tumor necrosis factor-α and the activation of AKT, nuclear factor-κB, and extracellular regulated kinases 1/2 by HMGB1 in HUVECs. Fisetin also down-regulated CLP-induced release of HMGB1, production of interleukin 1β, and reduced septic mortality. Collectively, these results suggest that fisetin may be a candidate therapeutic agent for the treatment of vascular inflammatory diseases via inhibition of the HMGB1 signaling pathway.     

In Vitro and In Vivo Characterization of MEMS Microneedles

Transdermal drug delivery TDD systems have many advantages but are conventionally limited by the low permeability of skin. The idea of using microneedles to painlessly penetrate the topmost impermeable stratum corneum has previously been put forward. In this paper, the fabrication of solid and hollow silicon microneedles with straight side-walls and with the following dimensions: 20–100 m in diameter and 100–150 m in length is described. In vitro tests demonstrate that with prior solid microneedle application, transdermal drug transport is significantly increased by 10–20 times, with the degree of enhancement being related to needle diameter. In vivo tests in diabetic animals, however, were unable to demonstrate any delivery of insulin through the hollow microneedles. It is proposed that two factors, microneedle length and tip sharpness, have to be improved for systemic drug delivery to be seen in vivo.     

Antigen-Pulsed, Interleukin-4-Treated B Cells Activate Primed T Cells In Vitro but not Naive T Cells In Vivo

The ability of B cells to act as effective antigen-presenting cells is a source of debate which centres on the degree of activation of either B cells or T cells. We have investigated whether B cells treated with interleukin 4 (IL-4) can express the two signals required to activate T cells: MHC Class 2/antigenic peptide complexes(signal 1) and the costimulatory molecules B7-1 and B7-2 (signal 2). We have also determined whether these cells could activate atitigen-experienced T cells in vitro and whether they could prime naive T cells in vivo . We found that B cells expressed abutidant MHC Class 2 molecules and moderate levels of B7-2 after 24 h culture in IL-4 with or without purified protein derivative (PPD) but B7-1 was not detectable. PPD-pulsed, IL-4 treated B cells induced antigeti-experienced T cells to proliferate in vitro but these cells failed to prime naive T cells in vivo when injected itito mice. We conclude that signals, in addition to those mduced with IL-4, are required for B cells to initiate an immutie respotise to atitigen.     

Murine Dendritic Cells Pulsed In Vitro with Toxoplasma gondii Antigens Induce Protective Immunity In Vivo

The activation of a predominant T-helper-cell subset plays a critical role in disease resolution. In the case of Toxoplasma gondii, the available evidence indicates that CD4⁺ protective cells belong to the Th1 subset. The aim of this study was to determine whether T. gondii antigens (in T. gondii sonicate [TSo]) presented by splenic dendritic cells (DC) were able to induce a specific immune response in vivo and to protect CBA/J mice orally challenged with T. gondii cysts. CBA/J mice immunized with TSo-pulsed DC exhibited significantly fewer cysts in their brains after oral infection with T. gondii 76K than control mice did. Protected mice developed a strong humoral response in vivo, with especially high levels of anti-TSo immunoglobulin G2a antibodies in serum. T. gondii antigens such as SAG1 (surface protein), SAG2 (surface protein), MIC1 (microneme protein), ROP2 through ROP4 (rhoptry proteins), and MIC2 (microneme protein) were recognized predominantly. Furthermore, DC loaded with TSo, which synthesized high levels of interleukin-12 (IL-12), triggered a strong cellular response in vivo, as assessed by the proliferation of lymph node cells in response to TSo restimulation in vitro. Cellular proliferation was associated with gamma interferon and IL-2 production. Taken together, these results indicate that immunization of CBA/J mice with TSo-pulsed DC can induce both humoral and Th1-like cellular immune responses and affords partial resistance against the establishment of chronic toxoplasmosis.     

Bordetella avium Virulence Measured In Vivo and In Vitro

Bordetella avium causes an upper-respiratory-tract disease called bordetellosis in birds. Bordetellosis shares many of the clinical and histopathological features of disease caused in mammals by Bordetella pertussis and Bordetella bronchiseptica. In this study we determined several parameters of infection in the domestic turkey, Meleagris galapavo, and compared these in vivo findings with an in vitro measure of adherence using turkey tracheal rings. In the in vivo experiments, we determined the effects of age, group size, infection duration, and interindividual spread of B. avium. Also, the effect of host genetic background on susceptibility was tested in the five major commercial turkey lines by infecting each with the parental B. avium strain and three B. avium insertion mutants. The mutant strains lacked either motility, the ability to agglutinate guinea pig erythrocytes, or the ability to produce dermonecrotic toxin. The susceptibilities of 1-day-old and 1-week-old turkeys to B. avium were the same, and challenge group size (5, 8, or 10 birds) had no effect upon the 50% infectious dose. Two weeks between inoculation and tracheal culture was optimal, since an avirulent mutant (unable to produce dermonecrotic toxin) persisted for a shorter time. Communicability of the B. avium parental strain between confined birds was modest, but a nonmotile mutant was less able to spread between birds. There were no host-associated differences in susceptibility to the parental strain and the three B. avium mutant strains just mentioned: in all turkey lines tested, the dermonecrotic toxin- and hemagglutination-negative mutants were avirulent whereas the nonmotile mutants showed no loss of virulence. Interestingly, the ability of a strain to cause disease in vivo correlated completely with its ability to adhere to ciliated tracheal cells in vitro.