Retinoic acid increases the effect of bone morphogenetic protein type 2 on osteogenic differentiation of human adipose-derived stem cells

Abstract Bone morphogenetic protein type 2 (BMP-2) and retinoic acid (RA) are osteoinductive factors that stimulate endogenous mechanisms of bone repair which can be applied on management of osseous defects in oral and maxillofacial fields. Objective Considering the different results of RA on osteogenesis and its possible use to substitute/potency BMP-2 effects, this study evaluated the outcomes of BMP-2, RA, and BMP-2+RA treatments on in vitro osteogenic differentiation of human adipose-derived stem cells (ASCs) and the signaling pathway(s) involved. Material and Methods ASCs were treated every other day with basic osteogenic medium (OM) alone or supplemented with BMP-2, RA, or BMP-2+RA. Alkaline phosphatase (ALP) activity was determined using the r-nitrophenol method. Extracellular matrix mineralization was evaluated using von Kossa staining and calcium quantification. Expression of osteonectin and osteocalcin mRNA were determined using qPCR. Smad1, Smad4, phosphorylated Smad1/5/8, BMP-4, and BMP-7 proteins expressions were analyzed using western blotting. Signaling pathway was evaluated using the IPA® software. Results RA promoted the highest ALP activity at days 7, 14, 21, and 28, in comparison to BMP-2 and BMP-2+RA. BMP-2+RA best stimulated phosphorylated Smad1/5/8 protein expression at day 7 and Smad4 expression at days 7, 14, 21, and 28. Osteocalcin and osteonectin mRNA expressions were best stimulated by BMP-2+RA at day 7. Matrix mineralization was most improved by BMP-2+RA at days 12 and 32. Additionally, BMP-2+RA promoted the highest BMP signaling pathway activation at days 7 and 14, and demonstrated more activation of differentiation of bone-forming cells than OM alone. Conclusions In summary, RA increased the effect of BMP-2 on osteogenic differentiation of human ASCs.

In vitro intestinal permeability studies, pharmacokinetics and tissue distribution of 6-methylcoumarin after oral and intraperitoneal administration in Wistar rats

ABSTRACT 6-Methylcoumarin (6MC) is a semisynthetic coumarin with important in vitro and in vivo anti-inflammatory activity. In order to continue the pre-clinical characterization of this molecule, in vitro intestinal permeability, plasma profile and tissue distribution after oral administration in rats were studied. The permeability of 6MC was evaluated by the Caco-2 cellular model in both the apical-basal (A-B) and basal-apical (B-A) directions. The pharmacokinetics and biodistribution were evaluated in rats after oral and intraperitoneal administration at doses of 200 mg/kg. Transport experiments with Caco-2 cells showed that 6MC presented high permeability at all concentrations evaluated. This finding suggested that 6MC could be transported across the gut wall by passive diffusion. The plasma concentration-time curve showed that the maximum concentration (Cmax) was 17.13 ± 2.90 µg/mL at maximum time (Tmax) of 30 min for the oral route and Cmax 26.18 ± 2.47 µg/mL at 6.0 min for the intraperitoneal administration, with elimination constant of (Ke ) 0.0070 min-1 and a short life half time of (T1/2 ) lower that 120 min. The distribution study showed that 6MC has high accumulation in the liver, and widespread distribution in all the organs evaluated.

Addition of bone morphogenetic protein type 2 to ascorbate and β-glycerophosphate supplementation did not enhance osteogenic differentiation of human adipose-derived stem cells

Bone morphogenetic protein type 2 (BMP-2) is a potent local factor, which promotes bone formation and has been used as an osteogenic supplement for mesenchymal stem cells. OBJECTIVES: This study evaluated the effect of a recombinant BMP-2 as well as the endogenous BMP-4 and BMP-7 in the osteogenic differentiation of adipose-derived stem cells (ASCs) in medium supplemented with ascorbate and β-glycerophosphate. MATERIAL AND METHODS: Human ASCs were treated with osteogenic medium in the presence (ASCs+OM+BMP-2) or absence (ASCs+OM) of BMP-2. The alkaline phosphatase (ALP) activity was determined and the extracellular matrix mineralization was evaluated by Von Kossa staining and calcium quantification. The expressions of BMP-4, BMP-7, Smad1, Smad4, and phosphorylated Smad1/5/8 were analyzed by western blotting. Relative mRNA expressions of Smad1, BMP receptor type II (BMPR-II), osteonectin, and osteocalcin were evaluated by qPCR. Results: ASCs+OM demonstrated the highest expression of BMP-4 and BMP-7 at days 21 and 7, respectively, the highest levels of BMPR-II mRNA expression at day 28, and the highest levels of Smad1 mRNA at days 14 and 28. ASCs+OM+BMP-2 demonstrated the highest levels of Smad1 mRNA expression at days 1, 7, and 21, the highest expression of Smad1 at day 7, the highest expression of Smad4 at day 14, the highest ALP activity at days 14 and 21, and expression of phosphorylated Smad1/5/8 at day 7. ASCs+OM and ASCs+OM+BMP2 showed similar ALP activity at days 7 and 28, similar osteonectin and osteocalcin mRNA expression at all time periods, and similar calcium depositions at all time periods. CONCLUSIONS: We concluded that human ASCs expressed endogenous BMP-4 and BMP-7. Moreover, the supplementation of ASCs with BMP-2 did not increase the level of osteogenic markers in the initial (ALP activity), intermediate (osteonectin and osteocalcin), or final (calcium deposition) phases, suggesting that the exogenous addition of BMP-2 did not improve the in vitro osteogenesis process of human ASCs.

The depuration dynamics of oysters (Crassostrea gigas) artificially contaminated with hepatitis A virus and human adenovirus

Within the country of Brazil, Santa Catarina is a major shellfish producer. Detection of viral contamination is an important step to ensure production quality and consumer safety during this process. In this study, we used a depuration system and ultraviolet (UV) disinfection to eliminate viral pathogens from artificially infected oysters and analysed the results. Specifically, the oysters were contaminated with hepatitis A virus (HAV) or human adenovirus type 5 (HAdV5). After viral infection, the oysters were placed into a depuration tank and harvested after 48, 72 and 96 h. After sampling, various oyster tissues were dissected and homogenised and the viruses were eluted with alkaline conditions and precipitated with polyethylene glycol. The oyster samples were evaluated by cell culture methods, as well as polymerase chain reaction (PCR) and quantitative-PCR. Moreover, at the end of the depuration period, the disinfected seawater was collected and analysed by PCR. The molecular assays showed that the HAdV5 genome was present in all of the depuration time samples, while the HAV genome was undetectable after 72 h of depuration. However, viral viability tests (integrated cell culture-PCR and immunofluorescence assay) indicated that both viruses were inactivated with 96 h of seawater recirculation. In conclusion, after 96 h of UV treatment, the depuration system studied in this work purified oysters that were artificially contaminated with HAdV5 and HAV.

Anti-HSV-1 and anti-HIV-1 activity of gallic acid and pentyl gallate

The synthetic n-alkyl esters of gallic acid (GA), also known as gallates, especially propyl, octyl and dodecyl gallates, are widely employed as antioxidants by food and pharmaceutical industries. The inhibitory effects of GA and 15 gallates on Herpes Simplex Virus type 1 (HSV-1) and Human Immunodeficiency Virus (HIV-1) replication were investigated here. After a preliminary screening of these compounds, GA and pentyl gallate (PG) seemed to be the most active compounds against HSV-1 replication and their mode of action was characterized through a set of assays, which attempted to localize the step of the viral multiplication cycle where impairment occurred. The detected anti-HSV-1 activity was mediated by the inhibition of virus attachment to and penetration into cells, and by virucidal properties. Furthermore, an anti-HIV-1 activity was also found, to different degrees. In summary, our results suggest that both compounds could be regarded as promising candidates for the development of topical anti-HSV-1 agents, and further studies concerning the anti-HIV-1 activity of this group of molecules are merited.

In vitro antiviral activity of antimicrobial peptides against herpes simplex virus 1, adenovirus, and rotavirus

Peptides with broad-spectrum antimicrobial activity, known as antimicrobial peptides, have been isolated from distinct organisms. This paper describes the in vitro evaluation of the cytotoxicity and antiviral activity of nine peptides with different structures and origins against herpes simplex virus type 1, human adenovirus respiratory strain, and rotavirus SA11. Most of the evaluated peptides presented antiviral activity but they were only active near cytotoxic concentrations. Nevertheless, these results seem promising, and further modifications on the peptide's structures may improve their selectivity and reduce their cytotoxicity.

Comparison between immunomagnetic separation, coupled with immunofluorescence, and the techniques of Faust et al. and of Lutz for the diagnosis of Giardia lamblia cysts in human feces

In the present study, the performance of Immunomagnetic Separation technique, coupled with Immunofluorescence (IMS-IFA), was compared with the FAUST et al. and Lutz parasitological techniques for the detection of Giardia lamblia cysts in human feces. One hundred and twenty-seven samples were evaluated by the three techniques at the same time showing a rate of cyst detection of 27.5 percent by IMS-IFA and 15.7 percent by both Faust et al. and Lutz techniques. Data analysis showed a higher sensitivity of IMS-IFA for the detection of G. lamblia cysts in comparison with the techniques of FAUST et al. and Lutz. The use of this methodology as a routine procedure enables the processing of many samples simultaneously, in order to increase recovery rate of G. lamblia cysts and reduce the time of sample storage.

Botanical (morphological, micrographic), chemical and pharmacological characteristics of Pfaffia species (Amaranthaceae) native to South Brazil

Some parameters for the quality control of P. glomerata and P. paniculata roots using their botanical and chemical characteristics are presented. it was also carried out an in vitro pharmacological screening to evaluate some biological properties of P. glomerata that could be related to its popular use as "tonic". Relating to biological assays, ethanolic extract from P. glomerata roots did not presented antiviral, antiproliferative, antifungal or MAO inhibitory activities. The cytotoxicity evaluation of P. glomerata determined that `IC IND. 50ï is >2,000 µg/mL. The main morphological and micrographic characteristics of P. glomerata and P. paniculata roots are described in this paper in order to aid in their unequivocal identification

Equivalência terapêutica entre medicamentos fitoterápicos / Therapeutical equivalence among phytotherapics drugs

O estabelecimento da equivalência terapêutica entre medicamentos fitoterápicos é um processo complexo, exigindo a definição detalhada de parâmetros específicos, antes de se iniciar o processo de padronização. A exemplo dos medicamentos sintéticos, a fitoequivalência deve garantir ao paciente e ao médico a mesma eficácia e segurança terapêuticas. O exemplo apresentado demonstra a importância da precisão dos resultados de estudos de fitoequivalência, e mostram que, apesar da semelhança química, são necessários estudos complementares para verificar a equivalência terapêutica, especialmente no que diz respeito à ausência de toxicidade. Em se tratando de medicamentos fitoterápicos, a análise da equivalência terapêutica, via de regra, deve ser realizada por meio dos ensaios clínicos com extratos de plantas, das quais se conhece a substância ativa ou o grupo de substâncias ativas, o que possibilita a realização de estudos de bioequivalência e, conseqüentemente, a emissão de conclusões fidedignas a respeito da sua fitoequivalência.

Screening fitoquimico preliminar de alguns vegetais utilizados na medicina popular do Sul do Brasil-parte II / Preliminary phytochemical screening of some vegetables used in the folk medicine of the South of Brazil-part II

Este trabalho e a segunda parte de um levantamento fitoquimico preliminar de varias plantas de uso na medicina popular do sul do Brasil, e tambem de uma experiencia didatica com iniciacao cientifica para os alunos. Foram analisados as seguintes especies vegetais: "Citrus aurantium" L. var. "sinensis" (larangeira), "Cuphea balsamona (sete-sangria), "Dodonaea viscosa" Jacq. (vassoura-vermelha), "Malva sylvestris L." (malva), "Matricaria chamomilla L." (camomila), "Passiflora alata Dryander" (maracuja), "Persea americana Miller" (abacateiro), "Pimpinella anisum L." (erva-doce), "Sambucus nigra L." (sabugueiro), "Stevia rebaudiana Bert." (estevia) e "Valeriana officinallis L." (valeriana).

O controle de qualidade de insumos vegetais e fitoterápicos na Faculdade de Farmácia da Universidade Federal do Rio Grande do Sul / Quality control of plant drugs in the Faculdade de Farmácia da Universidade Federal do Rio Grande do Sul

Säo relatadas as análises de insumos vegetais e preparaçöes fitoterápicas efetuadas no Setor de Controle de Qualidade da Faculdade de Farmácia no período 1984-1986. Os resultados obtidos säo discutidos em relaçäo ao desenvolvimento de controle de qualidade na indústria

Investigaçäo farmacológica do extrato aquoso de folhas/caules de Achyrocline satureioides (LAM.) DC., compositae (Marcela) / Pharmacological research of aquous extracts from leaves/stems of Achyrocline satureioides (LAM) DC., composital (Marcela)

Achyrocline satureioides (Lam.) DC., Compositae, vulgarmente conhecida como "marcela", é uma planta amplamente utilizada em medicina popular no sul do Brasil por suas propriedades digestivas, colagogas, antiespasmódicas, carminativas, eupépticas e emenagogas. O presente trabalho foi desenvolvido com o objetivo de analisar o extrato bruto das folhas/caules da A. satureioides (Lam.) DC., em diversos modelos experimentais. Os resultados obtidos evidenciam que o extrato aquoso das folhas/caules de A. satureioides (Lam.) DC. possui atividade colinolítica e miorrelaxante. Além disso, sugerem um efeito sedativo central quando testado nas doses de 250 e 500 mg/kg, via oral e i.p. Em conjunto, os resultados obtidos justificam o emprego do infuso de folhas/caules da "marcela" pela medicina popular