Pyrosequencing Unveils Cystic Fibrosis Lung Microbiome Differences Associated with a Severe Lung Function Decline

Chronic airway infection is a hallmark feature of cystic fibrosis (CF) disease. In the present study, sputum samples from CF patients were collected and characterized by 16S rRNA gene-targeted approach, to assess how lung microbiota composition changes following a severe decline in lung function. In particular, we compared the airway microbiota of two groups of patients with CF, i.e. patients with a substantial decline in their lung function (SD) and patients with a stable lung function (S). The two groups showed a different bacterial composition, with SD patients reporting a more heterogeneous community than the S ones. Pseudomonas was the dominant genus in both S and SD patients followed by Staphylococcus and Prevotella. Other than the classical CF pathogens and the most commonly identified non-classical genera in CF, we found the presence of the unusual anaerobic genus Sneathia. Moreover, the oligotyping analysis revealed the presence of other minor genera described in CF, highlighting the polymicrobial nature of CF infection. Finally, the analysis of correlation and anti-correlation networks showed the presence of antagonism and ecological independence between members of Pseudomonas genus and the rest of CF airways microbiota, with S patients showing a more interconnected community in S patients than in SD ones. This population structure suggests a higher resilience of S microbiota with respect to SD, which in turn may hinder the potential adverse impact of aggressive pathogens (e.g. Pseudomonas). In conclusion, our findings shed a new light on CF airway microbiota ecology, improving current knowledge about its composition and polymicrobial interactions in patients with CF.     

Immunochemical quantitation of enzymes in human diploid cell line WI-38

A quantitative immunochemical study was carried out of four enzymes, cathodal esterase, acid phosphatase, glucosaminidase and β-glucuronidase. In homogenates of the human diploid cell line WI-38, the relative amounts of the enzymes increased with the passage number of the culture, although great variation was found in later passages just before death of the culture.     

Glucose‐stimulated insulin release: Parallel perifusion studies of free and hydrogel encapsulated human pancreatic islets

To explore the effects immune‐isolating encapsulation has on the insulin secretion of pancreatic islets and to improve our ability to quantitatively describe the glucose‐stimulated insulin release (GSIR) of pancreatic islets, we conducted dynamic perifusion experiments with isolated human islets. Free (unencapsulated) and hydrogel encapsulated islets were perifused, in parallel, using an automated multi‐channel system that allows sample collection with high temporal resolution. Results indicated that free human islets secrete less insulin per unit mass or islet equivalent (IEQ) than murine islets and with a less pronounced first‐phase peak. While small microcapsules (d = 700 µm) caused only a slightly delayed and blunted first‐phase insulin response compared to unencapsulated islets, larger capsules (d = 1,800 µm) completely blunted the first‐phase peak and decreased the total amount of insulin released. Experimentally obtained insulin time‐profiles were fitted with our complex insulin secretion computational model. This allowed further fine‐tuning of the hormone‐release parameters of this model, which was implemented in COMSOL Multiphysics to couple hormone secretion and nutrient consumption kinetics with diffusive and convective transport. The results of these GSIR experiments, which were also supported by computational modeling, indicate that larger capsules unavoidably lead to dampening of the first‐phase insulin response and to a sustained‐release type insulin secretion that can only slowly respond to changes in glucose concentration. Bioartificial pancreas type devices can provide long‐term and physiologically desirable solutions only if immunoisolation and biocompatibility considerations are integrated with optimized nutrient diffusion and insulin release characteristics by design.     

Hyaluronate receptor CD44 is expressed by astrocytes in the adult chicken and in astrocyte cell precursors in early development of the chick spinal cord.

The role of the hyaluronate receptor, CD44, is well known in adult mammal astrocytes where it modulates neuron-glia interactions. However, no data exist regarding its expression in other vertebrates during their development. In order to detect the expression of CD44 in the chicken and its possible involvement in glial precursor migratory patterns during spinal cord development, a monoclonal antibody (MoAb) against the mammalian standard isoform, CD44-H, was used in immunohistochemical and immunoblot assays. With these methods, CD44 hyaluronate receptors were found on mature astrocyte membranes of adult chicken spinal cord. Astrocytes were identified using a MoAb against GFAP. During development, small clusters of CD44 labelled cells were seen lining the central canal starting from embryonic stage E10. These labelled cells were dispersed in the dorsal, lateral and ventral funiculi of the spinal cord in the subsequent stages. After stage E15, the CD44 labelled cells were identified as astrocytes because of their GFAP immunoreactivity. We conclude that CD44 receptors on immature astrocyte precursors should be considered as early astrocyte markers which have a possible role during cell migratory dispersal.     

Immunomodulatory strategies prevent the development of autoimmune emphysema

Background

The presence of anti-endothelial cell antibodies and pathogenic T cells may reflect an autoimmune component in the pathogenesis of emphysema. Whether immune modulatory strategies can protect against the development of emphysema is not known.

Methods

Sprague Dawley rats were immunized with human umbilical vein endothelial cells (HUVEC) to induce autoimmune emphysema and treated with intrathymic HUVEC-injection and pristane. Measurements of alveolar airspace enlargement, cytokine levels, immuno histochemical, western blot analysis, and T cell repertoire of the lung tissue were performed.

Results

The immunomodulatory strategies protected lungs against cell death as demonstrated by reduced numbers of TUNEL and active caspase-3 positive cells and reduced levels of active caspase-3, when compared with lungs from HUVEC-immunized rats. Immunomodulatory strategies also suppressed anti-endothelial antibody production and preserved CNTF, IL-1alpha and VEGF levels. The immune deviation effects of the intrathymic HUVEC-injection were associated with an expansion of CD4+CD25+Foxp3+ regulatory T cells. Pristane treatment decreased the proportion of T cells expressing receptor beta-chain, Vβ16.1 in the lung tissue.

Conclusions

Our data demonstrate that interventions classically employed to induce central T cell tolerance (thymic inoculation of antigen) or to activate innate immune responses (pristane treatment) can prevent the development of autoimmune emphysema.     

Correlation of C4ST-1 and ChGn-2 expression with chondroitin sulfate chain elongation in atherosclerosis

Subendothelial retention of lipoproteins by proteoglycans (PGs) is the initiating event in atherosclerosis. The elongation of chondroitin sulfate (CS) chains is associated with increased low-density lipoprotein (LDL) binding and progression of atherosclerosis. Recently, it has been shown that 2 Golgi enzymes, chondroitin 4-O-sulfotransferase-1 (C4ST-1) and chondroitin N-acetylgalactosaminyltransferase-2 (ChGn-2), play a critical role in CS chain elongation. However, the roles of C4ST-1 and ChGn-2 during the progression of atherosclerosis are not known. The aim of this study was to analyze the expression of C4ST-1 and ChGn-2 in atherosclerotic lesions in vivo and determine whether their expression correlated with CS chain elongation.Low-density lipoprotein receptor knockout (LDLr KO) mice were fed a western diet for 2, 4, and 8 weeks to stimulate development of atherosclerosis. The binding of LDL and CS PG in this mouse model was confirmed by chondroitinase ABC (ChABC) digestion and apolipoprotein B (apo B) staining. Gel filtration analysis revealed that the CS chains began to elongate as early as 2 weeks after beginning a western diet and continued as the atherosclerosis progressed. Furthermore, quantitative real-time polymerase chain reaction (qRT-PCR) showed that the mRNA levels of C4ST-1 and ChGn-2 increased after 8 weeks of this diet. In contrast, the mRNA levels of their homologs, C4ST-2 and ChGn-1, were unchanged. In addition, immunohistochemical analysis demonstrated that the expression of C4ST-1 and ChGn-2 appeared to have similar site-specific patterns and coincided with biglycan expression at the aortic root.Our results suggested that C4ST-1 and ChGn-2 may be involved in the elongation of CS chains in the arterial wall during the progression of atherosclerosis. Therefore, modulating their expression and activity might be a novel therapeutic strategy for atherosclerosis.     

De novo lipogenesis maintains vascular homeostasis through endothelial nitric-oxide synthase (eNOS) palmitoylation

Endothelial dysfunction leads to lethal vascular complications in diabetes and related metabolic disorders. Here, we demonstrate that de novo lipogenesis, an insulin-dependent process driven by the multifunctional enzyme fatty-acid synthase (FAS), maintains endothelial function by targeting endothelial nitric-oxide synthase (eNOS) to the plasma membrane. In mice with endothelial inactivation of FAS (FASTie mice), eNOS membrane content and activity were decreased. eNOS and FAS were physically associated; eNOS palmitoylation was decreased in FAS-deficient cells, and incorporation of labeled carbon into eNOS-associated palmitate was FAS-dependent. FASTie mice manifested a proinflammatory state reflected as increases in vascular permeability, endothelial inflammatory markers, leukocyte migration, and susceptibility to LPS-induced death that was reversed with an NO donor. FAS-deficient endothelial cells showed deficient migratory capacity, and angiogenesis was decreased in FASTie mice subjected to hindlimb ischemia. Insulin induced FAS in endothelial cells freshly isolated from humans, and eNOS palmitoylation was decreased in mice with insulin-deficient or insulin-resistant diabetes. Thus, disrupting eNOS bioavailability through impaired lipogenesis identifies a novel mechanism coordinating nutritional status and tissue repair that may contribute to diabetic vascular disease.     

A three-dimensional constitutive model for the stress relaxation of articular ligaments

A new nonlinear constitutive model for the three-dimensional stress relaxation of articular ligaments is proposed. The model accounts for finite strains, anisotropy, and strain-dependent stress relaxation behavior exhibited by these ligaments. The model parameters are identified using published uniaxial stress–stretch and stress relaxation data on human medial collateral ligaments (MCLs) subjected to tensile tests in the fiber and transverse to the fiber directions (Quapp and Weiss in J Biomech Eng Trans ASME 120:757–763, 1998; Bonifasi-Lista et al. in J Orthop Res 23(1):67–76, 2005). The constitutive equation is then used to predict the nonlinear elastic and stress relaxation response of ligaments subjected to shear deformations in the fiber direction and transverse to the fiber direction, and an equibiaxial extension. A direct comparison with stress relaxation data collected by subjecting human MCLs to shear deformation in the fiber direction is presented in order to demonstrate the predictive capabilities of the model.     

Methylation-capture and Next-Generation Sequencing of free circulating DNA from human plasma

Background

Free circulating DNA (fcDNA) has many potential clinical applications, due to the non-invasive way in which it is collected. However, because of the low concentration of fcDNA in blood, genome-wide analysis carries many technical challenges that must be overcome before fcDNA studies can reach their full potential. There are currently no definitive standards for fcDNA collection, processing and whole-genome sequencing. We report novel detailed methodology for the capture of high-quality methylated fcDNA, library preparation and downstream genome-wide Next-Generation Sequencing. We also describe the effects of sample storage, processing and scaling on fcDNA recovery and quality.

Results

Use of serum versus plasma, and storage of blood prior to separation resulted in genomic DNA contamination, likely due to leukocyte lysis. Methylated fcDNA fragments were isolated from 5 donors using a methyl-binding protein-based protocol and appear as a discrete band of ~180 bases. This discrete band allows minimal sample loss at the size restriction step in library preparation for Next-Generation Sequencing, allowing for high-quality sequencing from minimal amounts of fcDNA. Following sequencing, we obtained 37×10⁶-86×10⁶ unique mappable reads, representing more than 50% of total mappable reads. The methylation status of 9 genomic regions as determined by DNA capture and sequencing was independently validated by clonal bisulphite sequencing.

Conclusions

Our optimized methods provide high-quality methylated fcDNA suitable for whole-genome sequencing, and allow good library complexity and accurate sequencing, despite using less than half of the recommended minimum input DNA.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-476) contains supplementary material, which is available to authorized users.