Role of TRPA1 receptors in skin inflammation induced by volatile chemical irritants in mice.

Contact dermatitis is a very common inflammatory reaction in the skin, causing not only aesthetic problems but also loss functionality at work. The molecular mechanisms of contact dermatitis induced by chemical irritants are still unclear. Considering that transient receptor potential channels (TRP) may induce neurogenic inflammation and the exacerbation of inflammatory responses, here we investigated the role of transient receptor potential channel ankyrin type-1 (TRPA1) in skin inflammation evoked by chemical irritants. Ear oedema and nociceptive responses elicited by the topical application of xylene and toluene were measured in Swiss mice, wild type and TRPA1 knockout (Trpa1-/-) C57BL/6 mice. Histological analyses were performed in mice subjected to the ear oedema assay. Topical application of xylene and toluene in the mouse ear induced an edematogenic response (0.113 ±â€¯0.008 mm and 0.067 ±â€¯0.011 mm), compared to vehicle (0.008 ±â€¯0.008 mm), assessed by ear thickness measurements and histological analyses. These responses were prevented by topical pretreatment with a selective TRPA1 antagonist, HC-030031 (% inhibition: xylene 36.8 ±â€¯9.4% and toluene 50.7 ±â€¯11.0%), and by the genetic deletion of TRPA1 ((% inhibition: xylene 66.6 ±â€¯16.7% and toluene 75 ±â€¯0%). In addition, the topical application of xylene and toluene to the mouse paw elicited nociceptive responses, which were significantly reduced by oral treatment with HC-030031 ((% of inhibition: 84.9 ±â€¯1.3% and 27.1 ±â€¯8.0%, respectively); nociceptive responses were almost completely abolished in Trpa1-/-mice. Our data suggest that the activation of TRPA1 could be involved in some of the symptoms of irritant-mediated contact dermatitis, such as oedema, pain and neurogenic inflammation.

Uso de marcadores fluorescentes na engenharia tecidual óssea: estudo piloto / Use of fluorescent markers in bone tissue engineering: pilot study

Introdução: a regeneração e o reparo de tecidos ósseos perdidos é objeto de estudo da Bioengenharia Tecidual. O uso de biomateriais substitutos ósseos biomiméticos visa estimular os sistemas celulares e bioquímicos para restabelecer de modo mais eficiente o tecido ósseo nos casos de sua reconstrução. Ao investigar o processo de remodelação, é vital identificar áreas de novo crescimento para avaliar a eficácia dos biomateriais implantados e respectivos regimes de tratamento. A avaliação qualitativa e quantitativa da regeneração óssea pode ser realizada através da aplicação de marcadores como o Xilenol, a Tetraciclina, a Calceína e a Alizarina. A administração desses marcadores de forma associada possibilita ainda marcar sequencialmente camadas de nova deposição e remodelação durante o reparo. Objetivo: estabelecer um protocolo para utilização dos marcadores fluorescentes de reparo ósseo xilenol, tetraciclina, calceína e alizarina, em ratos. Metodologia: foram utilizados 35 ratos da linhagem Wistar, machos adultos, com massa corpórea entre 350 e 400g, e idade aproximada de 4 a 5 meses, distribuídos randomicamente em 5 grupos experimentais, submetidos à confecção de defeito ósseo circular de 8 mm em região de calvária, e administração dos diferentes marcadores segundo os grupos; XO ­ Xilenol; Ca ­ Calceína; Al ­ Alizarina; Te ­ Tetraciclina; C ­ Controle. Após 15 dias de experimento, os animais foram eutanasiados e as calvárias processadas e analisadas por histomorfometria, microscopia de epifluorescência e microscopia de fluorescência. Resultados: todos protocolos empregados para utilização dos marcadores fluorescentes xilenol, calceína, alizarina e tetracicilina foram úteis para identificar área de deposição mineral durante o período analisado de regeneração óssea em ratos. As imagens obtidas pela microscopia de fluorescência revela a presença dos marcadores incorporados à matriz óssea neoformada, no entanto a utilização da Alizarina e Calceína dentro dos protocolos testados mostraram-se mais eficientes. Conclusão: os protocolos testados nesse estudo apresentaram-se viáveis para utilização em pesquisas envolvendo marcadores de regeneração óssea, com resultados superiores para Alizarina e Calceína

Introduction: The regeneration and repair of lost bone tissues is the subject of a study of Tissue Bioengineering. The use of biomimetic biomaterial bone substitutes aims to stimulate the cellular and biochemical systems to restore more efficiently the bone tissue in the cases of its reconstruction. When investigating the remodeling process, it is vital to identify areas of new growth to evaluate the efficacy of implanted biomaterials and their treatment regimens. The qualitative and quantitative evaluation of bone regeneration can be performed through the use of markers such as Xylenol, Tetracycline, Calcein and Alizarin. The administration of such markers in an associated manner also makes it possible to sequentially mark layers of new deposition and remodeling during the repair. Objective: to establish a protocol for the use of fluorescent xylenol, tetracycline, calcein and alizarin bone repair markers in rats. Metodology: thirtyfive male adult Wistar rats with a body mass ranging from 350 to 400 g and approximately 4 to 5 months old were randomly assigned to 5 experimental groups submitted to a circular bone defect of 8 mm in the region of calvaria, and administration of the different markers according to the groups; XO ­ Xylenol; Ca ­ Calcein; Al-Alizarin; Te ­ Tetracycline; C ­ Control. After 15 days of experiment, the animals were euthanized and the calvaria processed and analyzed by histomorphometry, epifluorescence microscopy and fluorescence microscopy. Results: all protocols used for fluorescence markers xylenol, calcein, alizarin and tetracycline were useful to identify area of mineral deposition during the analyzed period of bone regeneration in rats. The images obtained by fluorescence microscopy revealed the presence of the markers incorporated into the neoformed bone matrix, however the use of Alizarin and Calcein within the protocols tested were more efficient. Conclusion: the protocols tested in this study were feasible for use in research involving markers of bone regeneration, with superior results for Alizarin and Calcein.

Parylene scaffold for cartilage lesion.

Evaluate parylene scaffold feasibility in cartilage lesion treatment, introducing a novel paradigm combining a reparative and superficial reconstructive procedure. Fifteen rabbits were used. All animals had both knees operated and the same osteochondral lesion model was created bilaterally. The parylene scaffold was implanted in the right knee, and the left knee of the same animal was used as control. The animals were euthanized at different time points after surgery: four animals at three weeks, three animals at six weeks, four animals at nine weeks, and four animals at 12 weeks. Specimens were analyzed by International Cartilage Repair Society (ICRS) macroscopic evaluation, modified Pineda histologic evaluation of cartilage repair, and collagen II immunostaining. Parylene knees were compared to its matched contra-lateral control knees of the same animal using the Wilcoxon matched-pairs signed rank. ICRS mean ± SD values for parylene versus control, three, six, nine and twelve weeks, respectively: 7.83 ± 1.85 versus 4.42 ± 1.08, p = 0.0005; 10.17 ± 1.17 versus 6.83 ± 1.17, p = 0.03; 10.89 ± 0.60 versus 7.33 ± 2.18, p = 0.007; 10.67 ± 0.78 versus 7.83 ± 3.40, p = 0.03. Modified Pineda mean ± SD values for parylene versus control, six, nine and twelve weeks, respectively: 3.37 ± 0.87 versus 6.94 ± 1.7, p < 0.0001; 5.73 ± 2.05 versus 6.41 ± 1.7, p = 0.007; 3.06 ± 1.61 versus 6.52 ± 1.51, p < 0.0001. No inflammation was seen. Parylene implanted knees demonstrated higher collagen II expression via immunostaining in comparison to the control knees. Parylene scaffolds are a feasible option for cartilage lesion treatment and the combination of a reparative to a superficial reconstructive procedure using parylene scaffolds led to better results than the reparative procedure alone.

Effects of musk ketone on nerve recovery after spinal cord injury.

The present study aimed to determine the effects of musk ketone on nerve recovery in rats after spinal cord injury. A total of 105 SD female rats were used to establish the rat with dorsal spinal cord injury model (modified Allen's method). The rats weighed from 200 to 250 g and were provided by the Experimental Animal Center of Chongqing Medical University. They were randomly divided into five treatment groups: saline (NS group), methylprednisolone (MP group), and musk ketone groups (MO1, MO2, and MO3 groups). The Swash plate test and BBB behavioral score were used to determine neurological function recovery after spinal cord injury. Hematoxylin-eosin (HE) staining was used to detect general structural changes in spinal cord tissue. The enzyme-linked immunosorbent assay was used for the determination of interleukin 10 (IL-10) in spinal cord tissue. We found that compared with the NS control group, critical angle, BBB score and IL-10 levels in rat spinal cord tissue significantly increased in the MP group and MO groups 7 and 14 days after the operation. HE staining showed that in the NS group, there was hemorrhage, edema, necrosis, axonal demyelination, inflammatory cell infiltration and glial cell response in spinal cord tissue. After 7 days, spinal cord edema and inflammation were reduced and neuronal degeneration and necrosis were not evident in the MP and MO groups. We conclude that musk ketone can reduce secondary damage after spinal cord injury and promote nerve recovery in rats.

Expression, production, and renaturation of a functional single-chain variable antibody fragment (scFv) against human ICAM-1

Intercellular adhesion molecule-1 (ICAM-1) is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19×10−8 M) against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv) was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35×10−7 M) by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.

Expression, production, and renaturation of a functional single-chain variable antibody fragment (scFv) against human ICAM-1.

Intercellular adhesion molecule-1 (ICAM-1) is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19 × 10(-8) M) against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv) was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35 × 10(-7) M) by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.

[Efficacy of biocides against hospital isolates of Staphylococcus sensitive and resistant to methicillin, in the province of Buenos Aires, Argentina]. / Eficacia de algunos biocidas contra estafilococos hospitalarios sensibles y resistentes a la meticilina en la provencia de Buenos Aires, Argentina.

OBJECTIVE: To assess the response to the action of different antiseptics and disinfectants usually used in Argentinian hospitals of hospital staphylococci sensitive and resistant to methicillin. To test the effectiveness of the biocides by measuring their effective bactericidal concentrations, and to determine whether there is any correlation between biocide resistance and methicillin resistance in this bacterial population. METHODS: The action of seven biocides was tested against 25 strains of nosocomial Staphylococcus spp. sensitive and resistant to methicillin, and in Staphylococcus aureus ATCC 6538. Hospital strains were obtained from April, 2000 to May, 2002, from clinical samples (blood culture, urine culture, catheter tip or abscess) from male and female inpatients and outpatients at two tertiary hospitals. After isolation, antibiotic sensitivity was tested with the agar diffusion method of Kirby and Bauer. The action of hospital biocides on the strains was studied with the Kelsey-Sykes test, which establishes the effective bactericide concentrations of these compounds. RESULTS: The results showed that the response of strains sensitive and resistant to methicillin varied in comparison to the collection strain. Chlorhexidine digluconate, povidone iodine, weak tincture of iodine and alkaline glutaraldehyde were effective against most strains, regardless of whether they were sensitive or resistant to methicillin. CONCLUSIONS: We found no indication of a relationship between resistance to methicillin and resistance to biocides. Our study shows that further research is needed to evaluate the efficacy of chemical agents against microorganisms that have been exposed to antibiotic therapies.

Quality control of decontaminating agents.

The present study evaluates the efficiency of the following decontaminating agents for the multiresistant, locally circulating bacterium Pseudomonas aeruginosa: glutaraldehyde 2%--makes A and B-, glutaraldehyde-formaldehyde; povidone-iodine-makes A, B and C-; sodium hypochloride; chloroxylenol--makes A and B-; and lapire chloride. The 9027 ATCC strain was used as a standard. A modification of the method of Kelsey and Sykes (1) was used to evaluate decontaminating efficiency. Highly satisfactory results were obtained with glutaraldehide 2% A and B, glutaraldehyde-formaldehyde and sodium hypochlorite. The results for povidone-iodine A, B and C were satisfactory but were unsatisfactory for chloroxylenol and lapirium chloride.