Reduction of the Inflammatory Responses against Alginate-Poly-L-Lysine Microcapsules by Anti-Biofouling Surfaces of PEG-b-PLL Diblock Copolymers

Large-scale application of alginate-poly-L-lysine (alginate-PLL) capsules used for microencapsulation of living cells is hampered by varying degrees of success, caused by tissue responses against the capsules in the host. A major cause is proinflammatory PLL which is applied at the surface to provide semipermeable properties and immunoprotection. In this study, we investigated whether application of poly(ethylene glycol)-block-poly(L-lysine hydrochloride) diblock copolymers (PEG-b-PLL) can reduce the responses against PLL on alginate-matrices. The application of PEG-b-PLL was studied in two manners: (i) as a substitute for PLL or (ii) as an anti-biofouling layer on top of a proinflammatory, but immunoprotective, semipermeable alginate-PLL₁₀₀ membrane. Transmission FTIR was applied to monitor the binding of PEG-b-PLL. When applied as a substitute for PLL, strong host responses in mice were observed. These responses were caused by insufficient binding of the PLL block of the diblock copolymers confirmed by FTIR. When PEG-b-PLL was applied as an anti-biofouling layer on top of PLL₁₀₀ the responses in mice were severely reduced. Building an effective anti-biofouling layer required 50 hours as confirmed by FTIR, immunocytochemistry and XPS. Our study provides new insight in the binding requirements of polyamino acids necessary to provide an immunoprotective membrane. Furthermore, we present a relatively simple method to mask proinflammatory components on the surface of microcapsules to reduce host responses. Finally, but most importantly, our study illustrates the importance of combining physicochemical and biological methods to understand the complex interactions at the capsules'' surface that determine the success or failure of microcapsules applicable for cell-encapsulation.     

Exploratory Activity of Rats in Three Different Environments

Adult male hooded (Long‐Evans) rats (Rattus norvergicus) were used to compare the efficiency of three types of tests (plain open field – OF, open field with a refuge – OD, and complex environment with a refuge – EX), for the evaluation of exploratory behaviour. The results confirmed that OF is a highly aversive situation with the animals showing elevated emotional response (defecation and urination) when compared to situations containing a refuge (OD and EX). Presence of a plain arena (OF and OD) does not elicit high exploratory performance, as shown by the delayed and reduced exit from the refuge in OD when compared to EX. Thus locomotor activity in OF (covered distance, number of rearings) probably reflects more of an escape reaction than a genuine exploratory behaviour. Conversely the presence of a complex environment seems to elicit a high exploratory performance expressed as a quick exit from the refuge, an increased time in the environment (more than 75% of the observation time) and intense locomotor patterns (large covered distances, great number of rearings) but with little emotional display (reduced defecation and urination). The results thus show the importance of considering ethological factors in the choice or development of laboratory tests.     

Phorbol esters induce nitric oxide synthase activity in rat hepatocytes. Antagonism with the induction elicited by lipopolysaccharide.

The incubation of primary cultures of rat hepatocytes with lipopolysaccharide (LPS) or biologically active phorbol esters promotes the release of nitric oxide to the incubation medium. This process is the result of the induction of the Ca(2+)-and calmodulin-independent form of nitric oxide synthase. Both the release of nitric oxide to the incubation medium and the expression of nitric oxide synthase activity exhibited a lag period of about 45-60 min after cell stimulation. Exposure of hepatocytes to both stimuli produced an antagonistic effect on nitric oxide release, with a half-maximal inhibition obtained with 14 nM phorbol 12,13-dibutyrate at saturating concentration of LPS. Incubation of cells with alpha-phorbol 12,13-didecanoate failed to counteract the effect of LPS or to induce nitric oxide synthase, suggesting that activation of protein kinase C was involved in this process.     

Spectral Sensitivity Measured with Electroretinogram Using a Constant Response Method

A new method is presented to determine the retinal spectral sensitivity function S(λ) using the electroretinogram (ERG). S(λ)s were assessed in three different species of myomorph rodents, Gerbils (Meriones unguiculatus), Wistar rats (Ratus norvegicus), and mice (Mus musculus). The method, called AC Constant Method, is based on a computerized automatic feedback system that adjusts light intensity to maintain a constant-response amplitude to a flickering stimulus throughout the spectrum, as it is scanned from 300 to 700 nm, and back. The results are presented as the reciprocal of the intensity at each wavelength required to maintain a constant peak to peak response amplitude. The resulting S(λ) had two peaks in all three rodent species, corresponding to ultraviolet and M cones, respectively: 359 nm and 511 nm for mice, 362 nm and 493 nm for gerbils, and 362 nm and 502 nm for rats. Results for mouse and gerbil were similar to literature reports of S(λ) functions obtained with other methods, confirming that the ERG associated to the AC Constant-Response Method was effective to obtain reliable S(λ) functions. In addition, due to its fast data collection time, the AC Constant Response Method has the advantage of keeping the eye in a constant light adapted state.     

NMR chiral discrimination of chalcogen containing secondary alcohols

Here, we report the general strategies by which NMR spectroscopy can be used to determine the enantiopurity and absolute configuration of chalcogen containing secondary alcohols, including the evaluation of the use of chiral solvating and chiral derivatizing agents. The BINOL/DMAP ternary complex demonstrated a simple and fast protocol for determining enantiopurity. The drug Naproxen afforded a stable, nonhygroscopic, and readily available chiral derivatizing agent (CDA) for NMR chiral discrimination of chalcogen containing secondary alcohols. The chiral recognition by CDA and chiral solvating agent (CSA) was assessed using ¹H, ⁷⁷Se‐{1H}, and ¹²⁵Te‐{1H} NMR spectroscopy. A simple model for the assignment of the absolute configuration from NMR data is presented.     

Proteomic characterization of Her2/neu-overexpressing breast cancer cells

The receptor tyrosine kinase HER2 is an oncogene amplified in invasive breast cancer and its overexpression in mammary epithelial cell lines is a strong determinant of a tumorigenic phenotype. Accordingly, HER2-overexpressing mammary tumors are commonly indicative of a poor prognosis in patients. Several quantitative proteomic studies have employed two-dimensional gel electrophoresis in combination with MS/MS, which provides only limited information about the molecular mechanisms underlying HER2/neu signaling. In the present study, we used a SILAC-based approach to compare the proteomic profile of normal breast epithelial cells with that of Her2/neu-overexpressing mammary epithelial cells, isolated from primary mammary tumors arising in mouse mammary tumor virus-Her2/neu transgenic mice. We identified 23 proteins with relevant annotated functions in breast cancer, showing a substantial differential expression. This included overexpression of creatine kinase, retinol-binding protein 1, thymosin 4 and tumor protein D52, which correlated with the tumorigenic phenotype of Her2-overexpressing cells. The differential expression pattern of two genes, gelsolin and retinol binding protein 1, was further validated in normal and tumor tissues. Finally, an in silico analysis of published cancer microarray data sets revealed a 23-gene signature, which can be used to predict the probability of metastasis-free survival in breast cancer patients.