Innate immune signalling at the intestinal epithelium in homeostasis and disease

The intestinal epithelium-which constitutes the interface between the enteric microbiota and host tissues-actively contributes to the maintenance of mucosal homeostasis and defends against pathogenic microbes. The recognition of conserved microbial products by cytosolic or transmembrane pattern recognition receptors in epithelial cells initiates signal transduction and influences effector cell function. However, the signalling pathways, effector molecules and regulatory mechanisms involved are not yet fully understood, and the functional outcome is poorly defined. This review analyses the complex and dynamic role of intestinal epithelial innate immune recognition and signalling, on the basis of results in intestinal epithelial cell-specific transgene or gene-deficient animals. This approach identifies specific epithelial cell functions within the diverse cellular composition of the mucosal tissue, in the presence of the complex and dynamic gut microbiota. These insights have thus provided a more comprehensive understanding of the role of the intestinal epithelium in innate immunity during homeostasis and disease.     

Virus chimeras for gene therapy, vaccination, and oncolysis: adenoviruses and beyond

Several challenges need to be addressed when developing viruses for clinical applications in gene therapy, vaccination, or viral oncolysis, including specific and efficient target cell transduction, virus delivery via the blood stream, and evasion of pre-existing immunity. With rising frequency, these goals are tackled by generating chimeric viruses containing nucleic acid fragments or proteins from two or more different viruses, thus combining different beneficial features of the parental viruses. These chimeras have boosted the development of virus-based treatment regimens for major inherited and acquired diseases, including cancer. Using adenoviruses as the paradigm and prominent examples from other virus families, we review the technological and functional advances in therapeutic virus chimera development and recent successful applications that can pave the way for future therapies.     

Modelling chlorophyll fluorescence of kiwi fruit (Actinidia deliciosa)

Kiwi fruit displays chlorophyll fluorescence. A physical model was developed to reproduce the observed original fluorescence for the whole fruit, from the emission of the different parts of the kiwi fruit. The spectral distribution of fluorescence in each part of the fruit, was corrected to eliminate distortions due to light re-absorption and it was analyzed in relation to photosystem II-photosystem I ratio. Kiwi fruit also displays variable chlorophyll-fluorescence, similar to that observed from leaves. The maximum quantum efficiency of photosystem II photochemistry (F(v)/F(m)), the quantum efficiency of photosystem II (Φ(PSII)), and the photochemical and non-photochemical quenching coefficients (q(P) and q(NP) respectively) were determined and discussed in terms of the model developed. The study was extended by determining the photosynthetic parameters as a function of the storage time, at both 4 °C and room temperature for 25 days.     

Expression and characterization of (15)N-labeled human C-reactive protein in Escherichia coli and Pichia pastoris for use in isotope-dilution mass spectrometry

Levels of C-reactive protein (CRP) in serum are correlated with inflammation and disease in humans. A higher level quantitative method, such as isotope-dilution mass spectrometry (ID-MS) is needed to compare and standardize the many commercial CRP assays. We compare the expression and purification of (15)N-CRP from Escherichia coli and Pichia pastoris and show that the protein isolated from P. pastoris has native pentameric structure along with high isotopic enrichment as shown by software developed specifically for this purpose. When this preparation was mixed in various ratios with unlabeled CRP and tryptic peptides of the mixtures were analyzed by LC-MS/MS, the ratios of heavy and light peaks were tightly correlated with input amounts of each protein. In this report we confirm the suitability of (15)N-rCRP as an internal standard in ID-MS. Standardization of CRP assays should help validate the relationship between CRP and human health.     

AICAR prevents heat-induced sudden death in RyR1 mutant mice independent of AMPK activation

Mice with a knock-in mutation (Y524S) in the type I ryanodine receptor (Ryr1), a mutation analogous to the Y522S mutation that is associated with malignant hyperthermia in humans, die when exposed to short periods of temperature elevation (≥37 °C). We show here that treatment with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) prevents this heat-induced sudden death in this mouse model. The protection by AICAR is independent of AMP-activated protein kinase (AMPK) activation and results from a newly identified action of the compound on mutant Ryr1 to reduce Ca(2+) leak from the sarcoplasmic reticulum to the sarcoplasm. AICAR thus prevents Ca(2+)-dependent increases in the amount of both reactive oxygen species (ROS) and reactive nitrogen species (RNS) that act to further increase resting Ca(2+) concentrations. If unchecked, the temperature-driven increases in resting Ca(2+) concentrations and the amounts of ROS and RNS create an amplifying cycle that ultimately triggers sustained muscle contractions, rhabdomyolysis and death. Although antioxidants are effective in reducing this cycle in vitro, only AICAR prevents heat-induced death in vivo. Our findings suggest that AICAR is probably effective in prophylactic treatment of humans with enhanced susceptibility to exercise- and/or heat-induced sudden death associated with RYR1 mutations.     

Lipid profile modifications in post-menopausal women treated with testosterone gel

ObjectiveTo assess lipid profile changes in post-menopausal women treated with testosterone gel.MethodsThirty-six oophorectomized women on estradiol treatment who received transdermal testosterone gel (5 mg daily) were enrolled into our study. Cholesterol, triglycerides, high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), very low density-lipoprotein cholesterol (VLDL-C), and lipoprotein (a) were tested before and after 6 months of treatment.ResultsSelected participants had a mean age of 50.9 ± 4.6 years and a body mass index of 30.1 ± 3.8 kg/m². Significantly decreased cholesterol levels were found after 6 months of treatment (204.5 ± 35.1 mg/dL before treatment as compared to 183.1 ± 21.9 mg/dL after treatment; p < 0.05). A significant reduction was also seen in LDL-C levels after 6 months of treatment with testosterone gel as compared to baseline (130.9 ± 29.7 mg/dL versus 118.5 ± 21.3 mg/dL; p < 0.05). No differences were found in triglyceride, HDL-C, VLDL-C, and lipoprotein (a) levels (p = ns).ConclusionEl gel de testosterona, asociado a tratamiento estrogénico en mujeres ooforectomizadas, produce disminución de las concentraciones de colesterol y LDL-C posterior a 6 meses de tratamiento, sin afectar las concentraciones de triglicéridos, HDL-C, VLDL-C y lipoproteína (a)Testosterone gel, associated to estrogen treatment in oophorectomized women, decreased cholesterol and LDL-C levels after 6 months of treatment, without affecting serum triglyceride, HDL-C, VLDL-C, and lipoprotein (a) levels.     

Non-native aggregation of recombinant human granulocyte-colony stimulating factor under simulated process stress conditions

Effective inhibition of protein aggregation is a major goal in biopharmaceutical production processes optimized for product quality. To examine the characteristics of process-stress-dependent aggregation of human granulocyte colony-stimulating factor (G-CSF), we applied controlled stirring and bubble aeration to a recombinant non-glycosylated preparation of the protein produced in Escherichia coli. We characterized the resulting denaturation in a time-resolved manner using probes for G-CSF conformation and size in both solution and the precipitate. G-CSF was precipitated rapidly from solutions that were aerated or stirred; only small amounts of soluble aggregates were found. Exposed hydrophobic surfaces were a characteristic of both soluble and insoluble G-CSF aggregates. Using confocal laser scanning microscopy, the aggregates presented mainly a circular shape. Their size varied according to incubation time and stress applied. The native intramolecular disulfide bonds in the insoluble G-CSF aggregates were largely disrupted as shown by mass spectrometry. New disulfide bonds formed during aggregation. All involved Cys(18) , which is the only free cysteine in G-CSF; one of them had an intermolecular Cys(18(A)) -Cys(18(B)) crosslink. Stabilization strategies can involve external addition of thiols and extensive reduction of surface exposition during processing.