17ß-estradiol replacement therapy induces eNOS, nNOS and estrogen receptor ß in hypophysectomized rats with inflamed footpads.

Nitric oxide (NO) plays a key role in inflammation. It is partly produced by three forms of NOS: eNOS of inflammatory cells, nNOS of neural cells and iNOS (inducible isoform). Estrogens can cause an anti-inflammatory effect, although it is not yet clear through which NOS isoforms. The aim of this study was to evaluate the role of the different NOS isoforms, as well as estrogen receptors (ERs) α and ß, on the anti-inflammatory effects of estrogens. To avoid the influence of endogenous glucocorticoids or sexual hormones, male rats were hypophysectomized. Animals were segregated into two control groups (no-treatment control group and SHAM-operated animals) and three hypophysectomized groups (no-hormonal treatment, with estradiol-17ß, or with testosterone replacement treatment). Freund's complete adjuvant (1 mg) was administered to the footpad of all animals. Measurements were made based on footpad inflammation (with a plethysmometer) such as eNOS, nNOS, iNOS and ER α and ß protein expression (by immunohistochemistry principle/method) on days 1, 7 and 14. Only estradiol decreased inflammation, accompanied by increased levels of eNOS and nNOS and differential expression of ERs α and ß in the inflammatory infiltrate. The higher levels of estradiol-induced eNOS and nNOS ocurred perhaps through the activation of ER ß.

Medicina Regenerativa para Úlceras Crónicas: Aplicación Cutánea de Células Troncales Mesenquimales de Gelatina de Wharton / Regenerative Medicine for Chronic Ulcers: Cutaneous Application of Mesenchymal Stem Cells of Wharton's Jelly

Resumen El empleo de células mesenquimales ha emergido como una parte importante en la medicina regenerativa con efectos positivos en la recuperación de tejidos dañados o perdidos. Las úlceras crónicas incluyen lesiones cutáneas que afectan a una gran parte de la población con diabetes mellitus, (por ejemplo 9.14% desarrollan lesiones por pie diabético según ENSANUT 2016) y representan gastos económicos considerables, debido a la pobre calidad de vida que llevan los pacientes. El tratamiento convencional que se lleva a cabo en estos casos es a largo plazo, y los efectos benéficos generados que se presentan son limitados, ya que las lesiones reinciden por no contar con un tratamiento específico y regenerativo, solo paliativo. Es por ello que en este trabajo se obtuvieron y emplearon células troncales mesenquimales derivadas de gelatina de Wharton (CTM-GW) bajo los criterios propuestos por la Sociedad Internacional de la Terapia Celular. El tratamiento fue aplicado en dos pacientes con úlceras crónicas flebostáticas de diferentes condiciones médicas y se monitoreo a corto plazo, por lo que fue un estudio limitado. Los resultados mostraron que las CTM-GW indujeron la reconstrucción cutánea mediante formación de tejido de granulación, además de disminuir el proceso inflamatorio crónico en la zona de daño, lo que favoreció el índice de cierre de la herida. Por lo anterior, se propone la utilización de CTM-GW en úlceras flebostáticas crónicas como tratamiento en la regeneración de la estructura tisular con resultados a corto plazo.

Abstract Use of mesenchymal cells has emerged as an important part in regenerative medicine with positive effects in the recovery of damaged or lost tissues. Chronic ulcers include skin lesions that affect a large part of the population with diabetes mellitus, (for example, 9.14 develop diabetic foot according to ENSANUT 2016) and represent considerable economic expenses, due to poor quality of life in patients. The conventional treatment that is carried out in these cases is long term, and the generated beneficial effects that are presented are limited, since the injuries recur due to not having a specific and regenerative treatment, only palliative. That is why in this work they were obtained and used mesenchymal stem cells derived from Wharton's jelly (MSC-WJ) under the criteria proposed by the International Society for Cell Therapy. The treatment was applied in two patients with chronic ulcers phlebostatic of different medical conditions and short-term monitoring, so it was a limited study. The results showed that MSC-WJ induced skin reconstruction by tissue formation granulation, in addition to decreasing the chronic inflammatory process in the area of damage, which favored the index of wound healing. Therefore, the use of MSC-WJ in chronic phlebostatic ulcers is proposed as a treatment in the regeneration of the tissue structure with short-term results.

Botulinum neurotoxin type A inhibits synaptic vesicle 2 expression in breast cancer cell lines.

AIM: It is known that botulinum neurotoxin type A (BoNTA) improves some kinds of cancer (e.g. prostate) and that synaptic vesicle glycoprotein 2 (SV2) is the molecular target of this neurotoxin. Besides having potential therapeutic value, this glycoprotein has recently been proposed as a molecular marker for several types of cancer. Although the mechanisms of cancer development and the improvement found with botulinum treatment are not well understood, the formation of the botulinum-SV2 complex may influence the presence and distribution of SV2 and the function of vesicles. To date, there are no reports on the possible effect of botulinum on breast cancer of unknown causes, which have a great impact on women's health. Thus we determined the presence of SV2 in three breast cancer cell lines and the alterations found with botulinum application. MATERIALS AND METHODS: With and without adding 10 units of botulinum, SV2 protein expression was determined by optical densitometry in T47D, MDA-MB-231 and MDA-MB-453 cell lines and the distribution of SV2 was observed with immunochemistry (hematoxylin staining). RESULTS: The SV2 protein was abundant in the cancer cells herein tested, and maximally so in T47D. In all three cancer cell lines botulinum diminished SV2 expression, which was found mostly in the cell periphery. CONCLUSION: SV2 could be a molecular marker in breast cancer. Its expression and distribution is regulated by botulinum, suggesting an interesting control mechanism for SV2 expression and a possible alternative therapy. Further studies are needed in this sense.

First Report of Neofusicoccum australe and N. luteum Associated with Canker and Dieback of Quercus robur in Portugal.

Quercus robur L., commonly known as "pedunculate oak," is a long-lived deciduous tree native to most of Europe. It is of great ecological and forestry importance. It is also commonly cultivated as an ornamental tree in parks and gardens. Since 2009 and most likely related to increased drought periods, diseased ornamental trees have been observed in the campus of the University of Aveiro, Portugal. More than 50% of the trees are already damaged by the disease. The symptoms included twig and branch dieback and sunken necrotic bark lesions that could progress to the trunk, resulting in the death of large sections of the tree. Ascomata and conidiomata typical of Botryosphaeriaceae were observed on branches of symptomatic trees. Ascospores were hyaline, aseptate, ovoid to fusoid, and conidia were hyaline, aseptate, smooth, thin-walled, and fusiform with base truncate. Single spore isolates were obtained from samples. In culture, single ascospore isolates produced conidia similar to the ones found on the host. Diseased branch tissues were surface sterilized with 5% NaOCl, plated on potato dextrose agar (PDA), and incubated at 25°C. Fungal isolates recovered produced white aerial mycelium that darkened with age becoming grey to dark grey and conidia that were similar in all aspects to the ones produced by single spore isolates. All isolates produced on PDA a yellow pigment that diffused into the agar and disappeared after 6 to 7 days. Morphological and cultural aspects of the isolates were similar to the species Neofusicoccum luteum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips and N. australe (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips (2). Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR fingerprinting divided the isolates into two groups (1). Two isolates (CAA352 and CAA392), one from each group, were selected for further identification by sequencing of the internal transcribed spacer (ITS) region of the rDNA and part of the elongation factor 1-alpha gene (EF1-α) (Accession No. ITS: JX975212 JX975213; EF1-α: JX975210 JX975211). BLAST analysis showed that ITS and EF1-α sequences from group 1 and 2 had 99 to 100% similarity to reference cultures (including ex-type) of N. luteum and N. australe, respectively. To confirm pathogenicity and fulfill Koch's postulates, six 2-year-old seedlings of Q. robur were artificially infected with isolates CAA352 and CAA392 and kept at approximately 20 to 25°C. A shallow wound was done with a scalpel on the basal part of the stem of each seedling, a bark portion was removed aseptically and a PDA disc (0.5 cm) of an actively growing culture was placed on the wound. Control seedlings received sterile PDA discs. The inoculation site was wrapped in Parafilm to prevent desiccation. Within 8 weeks, infected seedlings developed canker lesions associated with vascular necrosis around the inoculation point. A third of the seedlings died and developed abundant pycnidia on the stem. Control seedlings remained symptomless. Both pathogens were successfully reisolated from the infected tissue. N. luteum and N. australe are increasingly reported as causing diseases to a wide range of woody hosts of economic and forestry importance (3). To our knowledge, this is the first report of both species causing dieback and canker disease on Q. robur. References: (1) A. Alves et al. Res. Microbiol. 158:112, 2007. (2) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (3) B. Slippers et al. Fungal Biol. Rev. 21:90, 2007.

Synaptic vesicle protein 2 (SV2) isoforms.

New molecular markers of cancer had emerged with novel applications in cancer prevention and therapeutics, including for breast cancer of unknown causes, which has a high impact on the health of women worldwide. The purpose of this research was to determine protein and mRNA expression of synaptic vesicle 2 (SV2) isoforms A, B and C in breast cancer cell lines. Cultured cell lines MDA-MB-231, SKBR3, T47D were lysed and their protein and mRNA expression analyzed by real-time PCR and western blot technique, respectively. SV2A, B proteins were identified in non-tumor (MCF-10A) and tumor cell lines (MDA-MB-231 and T47D) while SV2C only was found in the T47D cell line. Furthermore, the genomic expression was consistent with protein expression for a such cell line, but in MDA-MB-231 there was no SV2B genomic expression, and the SV2C mRNA and protein were not found in the non tumoral cell line. These findings suggest a possible cellular transdifferentiation to neural character in breast cancer, of possible relevance to cancer development, and point to possible use of SV2 as molecular marker and a vehicle for cancer treatment with botulinum toxin.

High prevalence of ST121 in community-associated methicillin-susceptible Staphylococcus aureus lineages responsible for skin and soft tissue infections in Portuguese children.

In order to evaluate the incidence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in Portugal, we analyzed a collection of 38 S. aureus isolates recovered from 30 children attending the pediatric emergency department of a central hospital in Lisbon due to skin and soft tissue infections. Molecular characterization identified seven clonal lineages among the 35 methicillin-susceptible S. aureus (MSSA) isolates, of which the major lineage PFGE A/t159/ST121 included 63% of the isolates. The three MRSA isolates belonged to the Pediatric clone PFGE D/t535/ST5-IV (n = 2) and to the European CA-MRSA clone PFGE G/t044/ST80-IVc (n = 1). All isolates harbored several virulence factors, namely, leukocidins. Panton-Valentine leukocidin (PVL) was produced by isolates from five MSSA lineages and by the ST80 MRSA. Of interest, this is the first reported isolation of CA-MRSA ST80 in Portugal.

Composted municipal waste effects on chemical properties of a Brazilian soil.

The spread of composted municipal waste (CMW) on land can be used for sustainable crop production. Nevertheless, heavy metals availability may be a problem. Therefore, the main objective of this study was to assess the impact of CMW disposal on heavy metal accumulation in soil and plants. The treatments consisted of an untreated plot (control) and four rates of CMW application. All plots were cultivated in succession of carrot, cauliflower, sweet corn, and radish. Cu and Pb significantly accumulated in the topsoil (0-5 cm) with a similar pattern in the depths of 5-10 cm and 10-20 cm. Cauliflower, for Fe and Cu, and radish, for Pb and Cu, had their tissue analysis significantly affected due to the increasing rates of application of CMW. Nevertheless, the levels of accumulation in both, soil and plant, are within permissible limits. The evidences provided by this experiment indicated that heavy metals are less likely to cause problems for the estimation of CMW loadings to Brazilian agricultural land.

IgG subclasses serum concentrations in a population of children with Down syndrome: comparative study with siblings and general population.

Serum total IgG and subclasses were determined in three different groups of children: with Down syndrome, their siblings and general pediatric population. Several cases of IgG2 and IgG4 deficiency were identified, predominantly in children with Down syndrome. The differences, considering three age groups, were statistically significant for both groups in relation to the general population group, with an increase of IgG1 and IgG3 and a decrease in serum concentrations of IgG2 and IgG4. Down syndrome children and their siblings tend o have a similar variation of the IgG4 serum concentration levels (P < 0.05). The mechanisms of this concordance are not well understood. The results point out that an adequate strategy to improve the immune status of Down syndrome children could have a positive manifestation in the immune profile of their brothers.

IgG subclasses serum concentrations in a population of children with Down syndrome: Comparative study with siblings and general population

Serum total IgG and subclasses were determined in three different groups of children: with Down syndrome, their siblings and general pediatric population. Several cases of IgG2 and IgG4 deficiency were identified, predominantly in children with Down syndrome. The differences, considering three age groups, were statistically significant for both groups in relation to the general population group, with an increase of IgG1 and IgG3 and a decrease in serum concentrations of IgG2 and IgG4. Down syndrome children and their siblings tend o have a similar variation of the IgG4 serum concentration levels (P < 0.05). The mechanisms of this concordance are not well understood. The results point out that an adequate strategy to improve the immune status of Down syndrome children could have a positive manifestation in the immune profile of their brothers (AU)

Se determinaron el suero total IgG y subclases en tres grupos distintos de niños: con síndrome de Down, sus hermanos y población pediátrica general. Se identificaron varios casos de deficiencia en suero IgG2 e IgG4, principalmente en niños con síndrome de Down. Las diferencias, considerando tres grupos de edad, resultaban estadísticamente significativas en ambos grupos en relación con el grupo de población general, con un aumento de IgG1 e IgG3 y un descenso de las concentraciones de suero de IgG2 e IgG4. Los niños con síndrome de Down y sus hermanos tienen tendencia a presentar una variación similar de los niveles de concentración de suero IgG4 (p < 0,05). Los mecanismos de esta concordancia no se entienden demasiado bien. Los resultados indican que una estrategia adecuada para mejorar las condiciones inmunológicas de los niños con síndrome de Down podría tener una manifestación positiva en el perfil inmunológico de sus hermanos (AU)

Na(+)/K(+)-ATPase activity and immunocytochemical labeling in podobranchial filament and lamina of the freshwater crayfish Astacus leptodactylus Eschscholtz: evidence for the existence of sodium transport in the filaments.

In the crayfish Astacus leptodactylus, the podobranch, bearing respiratory and ion-transporting filaments, is attached to the lamina. The Na(+)/K(+)-ATPase activity is higher in filaments than in lamina. Using a mouse monoclonal antibody directed against the alpha subunit of Na(+)/K(+)-ATPase, and indirect immunofluorescence microscopy, we can observe that the immunoreactivity of the antibody is different for each kind of structure. The lamina presents an ion-transporting type epithelium, which is thick and presents a developed apical infolding system but no developed basolateral infolding system. The immunoreactivity of the antibody in the lamina is very weak. Respiratory filaments present a thin epithelium with a few small apical folds and scarce mitochondria. The immunoreactivity of the antibody in the respiratory filaments is very weak. In contrast, the ion-transporting filament epithelium is thick, presents a short apical infolding system, a well-developed basal infolding system, and numerous mitochondria. The immunoreactivity of the antibody in the ion-transporting filaments is strong. Ion-transporting filaments, which have a cuticle permeable to cations, seem particularly implicated in Na(+)regulation, by way of the sodium pump.

Transepithelial potential difference of a single gill filament isolated from the crayfish Astacus leptodactylus Esch.: a new method.

A new method is described that allows in vitro perfusion and transepithelial electrical potential measurements of a single filament (3-5 mm long; 200 microns in diameter) isolated from the podobranch of the crayfish Astacus leptodactylus. An electrophysiological study was carried out on the preparation to validate this technique. The good physiological quality of the isolated filament preparation has been established and results of continuous measurements of the potential difference under two perfusion conditions are reported. Filaments were perfused with Van Harreveld physiological saline inside and either with Van Harreveld saline or artificial fresh water outside. Large potential differences up to 150 mV between inside and outside of the filament were recorded. When filaments were symmetrically perfused, the behavior of the electrical potential difference allowed two populations of filaments to be distinguished.