Sponge-mediated lentivirus delivery to acute and chronic spinal cord injuries.

The environment within the spinal cord after injury, which changes in the progression from the acute to chronic stages, limits the extent of regeneration. The delivery of inductive factors to promote regeneration following spinal cord injury has been promising, yet, few strategies are versatile to allow delivery during acute or chronic injury that would facilitate screening of candidate therapies. This report investigates the intrathecal delivery of lentiviruses for long-term expression of regenerative factors. Lentivirus-filled sponges were inserted into the intrathecal space surrounding the spinal cord, with transgene expression observed within multiple cell types that persists for 12 weeks for both intact and injured spinal cord, without any apparent damage to the spinal cord tissue. Sponges loaded with lentivirus encoding for Sonic hedgehog (Shh) were investigated for acute (delivered at 0 weeks) and chronic (at 4 weeks) injuries, and for multiple locations relative to the injury. In an acute model, sponges placed directly above the injury increased oligodendrocyte and decreased astrocyte presence. Sponges placed caudal to the injury had reduced impact on oligodendrocytes and astrocytes in the injury. In a chronic model, sponges increased oligodendrocyte and decreased astrocyte presence. Furthermore, the effect of Shh was shown to be mediated in part by reduction of Bmp signaling, monitored with an Msx2-sensitive reporter vector. The implantation of lentivirus-loaded biomaterials intrathecally provides the opportunity to induce the expression of a factor at a specified time without entering the spinal cord, and has the potential to promote gene delivery within the spinal cord, which can influence the extent of regeneration.

Heparin-chitosan nanoparticle functionalization of porous poly(ethylene glycol) hydrogels for localized lentivirus delivery of angiogenic factors.

Hydrogels have been extensively used for regenerative medicine strategies given their tailorable mechanical and chemical properties. Gene delivery represents a promising strategy by which to enhance the bioactivity of the hydrogels, though the efficiency and localization of gene transfer have been challenging. Here, we functionalized porous poly(ethylene glycol) hydrogels with heparin-chitosan nanoparticles to retain the vectors locally and enhance lentivirus delivery while minimizing changes to hydrogel architecture and mechanical properties. The immobilization of nanoparticles, as compared to homogeneous heparin and/or chitosan, is essential to lentivirus immobilization and retention of activity. Using this gene-delivering platform, we over-expressed the angiogenic factors sonic hedgehog (Shh) and vascular endothelial growth factor (Vegf) to promote blood vessel recruitment to the implant site. Shh enhanced endothelial recruitment and blood vessel formation around the hydrogel compared to both Vegf-delivering and control hydrogels. The nanoparticle-modified porous hydrogels for delivering gene therapy vectors can provide a platform for numerous regenerative medicine applications.

Sensitive PCR-based quantitation of cell-free circulating microRNAs.

Cell-free microRNAs (miRNAs) that circulate in the blood are promising surrogate biomarkers of disease and physiological processes. The ease of quantifying specific miRNA species using made-to-order approaches based on Taq-polymerase has led to numerous studies that have identified changes in the abundance of circulating cell-free miRNA species that correlate with pathology or other events. The growing interest in developing miRNAs as blood biomarkers necessitates the careful consideration of the unique properties of such body fluids that can make the reproducible and quantitative assessment of RNA abundance challenging. For example, enzymes involved in the amplification and analysis of RNA can be affected by blood components that copurify with miRNA. Thus, if miRNAs are to be effectively utilized as biomarkers, it is important to establish standardized protocols for blood collection and miRNA analysis to ensure accurate quantitation. Here we outline several considerations, including the type of collection tube used in sampling, the influence of added anticoagulants and stabilizers, sample processing, enrichment of vesicular and other miRNA species, RNA extraction approaches and enzyme selection, that affect quantitation of miRNA isolated from plasma and should be considered in order to achieve reproducible, sensitive and accurate quantitation.

MicroRNAs are exported from malignant cells in customized particles.

MicroRNAs (miRNAs) are released from cells in association with proteins or microvesicles. We previously reported that malignant transformation changes the assortment of released miRNAs by affecting whether a particular miRNA species is released or retained by the cell. How this selectivity occurs is unclear. Here we report that selectively exported miRNAs, whose release is increased in malignant cells, are packaged in structures that are different from those that carry neutrally released miRNAs (n-miRNAs), whose release is not affected by malignancy. By separating breast cancer cell microvesicles, we find that selectively released miRNAs associate with exosomes and nucleosomes. However, n-miRNAs of breast cancer cells associate with unconventional exosomes, which are larger than conventional exosomes and enriched in CD44, a protein relevant to breast cancer metastasis. Based on their large size, we call these vesicles L-exosomes. Contrary to the distribution of miRNAs among different microvesicles of breast cancer cells, normal cells release all measured miRNAs in a single type of vesicle. Our results suggest that malignant transformation alters the pathways through which specific miRNAs are exported from cells. These changes in the particles and their miRNA cargo could be used to detect the presence of malignant cells in the body.

Plasma components affect accuracy of circulating cancer-related microRNA quantitation.

Circulating microRNAs (miRNAs) have emerged as candidate biomarkers of various diseases and conditions including malignancy and pregnancy. This approach requires sensitive and accurate quantitation of miRNA concentrations in body fluids. Herein we report that enzyme-based miRNA quantitation, which is currently the mainstream approach for identifying differences in miRNA abundance among samples, is skewed by endogenous serum factors that co-purify with miRNAs and anticoagulant agents used during collection. Of importance, different miRNAs were affected to varying extent among patient samples. By developing measures to overcome these interfering activities, we increased the accuracy, and improved the sensitivity of miRNA detection up to 30-fold. Overall, the present study outlines key factors that prevent accurate miRNA quantitation in body fluids and provides approaches that enable faithful quantitation of miRNA abundance in body fluids.

Nuevos antecedentes sobre estrongiloidiasis humana en Arica / Human strongyloidiasis in Arica

Se analiza la frecuencia de estrongiloidiasis en un grupo de 25.002 pacientes ambulatorios y hospitalizados de la ciudad de Arica. El diagnóstico se realizó mediante el hallazgo de larvas rabditoides de Strongyloides stercoralis en el examen coproparasitológico seriado. De dicho número, 20 pacientes (0,08%) presentaron las larvas del nemátodo en sus deposiciones. No se observaron diferencias significativas en relación al sexo (11 hombres y 9 mujeres), y a la edad (10 niños y 10 adultos). Se pudo revisar la historia clínica de 10 casos, observándose una eosinofilia promedio de 23,35%, (fluctuante entre 10 y 34%). El 60% presentaba además otros parásitos y/o comensales asociados

Estrongiloidiasis en Arica / Strongyloidiasis in Arica

The presence of Stongyloides stercoralis in the faexes of 20 persons from Arica, Chile is reported