High Throughput Sequencing Analysis of the Immunoglobulin Heavy Chain Gene from Flow-Sorted B Cell Sub-Populations Define the Dynamics of Follicular Lymphoma Clonal Evolution

Understanding the dynamics of evolution of Follicular Lymphoma (FL) clones during disease progression is important for monitoring and targeting this tumor effectively. Genetic profiling of serial FL biopsies and examples of FL transmission following bone marrow transplant suggest that this disease may evolve by divergent evolution from a common ancestor cell. However where this ancestor cell resides and how it evolves is still unclear. The analysis of the pattern of somatic hypermutation of the immunoglobulin gene (Ig) is traditionally used for tracking the physiological clonal evolution of B cells within the germinal center and allows to discriminate those cells that have just entered the germinal center and display features of ancestor cells from those B cells that keep re-circulating across different lymphoid organs. Here we investigated the pattern of somatic hypermutation of the heavy chain of the immunoglobulin gene (IgH-VH) in 4 flow-sorted B cells subpopulations belonging to different stages of differentiation, from sequential lymph node biopsies of cases displaying diverse patterns of evolution, using the GS-FLX Titanium sequencing platform. We observed an unexpectedly high level of clonality, with hundreds of distinct tumor subclones in the different subpopulations from the same sample, the majority detected at a frequency <10⁻². By using a lineage trees analysis we observed in all our FL and t-FL cases that the oligoclonal FL population was trapped in a narrow intermediate stage of maturation that maintains the capacity to undergo SHM, but was unable to further differentiate. The presence of such a complex architecture highlights challenges currently encountered in finding a cure for this disease.     

Chlorophyll fluorescence quenching in the alga Euglena gracilis

When far red light preincubated cells of Euglena gracilis are transferred to dark or light, chlorophyll fluorescence (F₀ and F_m) decreases. Non-photochemical quenching in the dark is suggested to be induced partly by chlororespiration and partly by changes in the distribution of excitation energy between the photosystems. Depending on the light intensities it was possible to resolve the non-photochemical quenching into at least three different components. The slowest relaxation phase of non-photochemical quenching occurred only after exposure to high light and was assigned to photoinhibition. The other two components were an energy-dependent quenching (qE), and the one which we attribute to a spill over mechanism. We suggest that both photosystems use a common antenna system consisting of LHC I and LHC II proteins. In contrast to higher plants, qE in Euglena gracilis is independent of the xanthophyll cycle and an aggregation of LHC II. This revised version was published online in June 2006 with corrections to the Cover Date.     

The antimicrobial effect of Octenidine-dihydrochloride coated polymer tracheotomy tubes on Staphylococcus aureus and Pseudomonas aeruginosa colonisation

Background  

The surface of polymeric tracheotomy tubes is a favourable environment for biofilm formation and therefore represents a potential risk factor for the development of pneumonia after tracheotomy. The aim of this in-vitro study was to develop octenidine-dihydrochloride (OCT) coated polymer tracheotomy tubes and investigate any effects on Staphylococcus (S.) aureus and Pseudomonas (P.) aeruginosa colonization. Additionally the resistance of the OCT coating was tested using reprocessing procedures like brushing, rinsing and disinfection with glutaraldehyde     

People with multiple tattoos and/or piercings are not at increased risk for HBV or HCV in The Netherlands

Background

Although published results are inconsistent, it has been suggested that tattooing and piercing are risk factors for HBV and HCV infections. To examine whether tattooing and piercing do indeed increase the risk of infection, we conducted a study among people with multiple tattoos and/or piercings in the Netherlands who acquired their tattoos and piercings in the Netherlands and/or abroad.

Methods

Tattoo artists, piercers, and people with multiple tattoos and/or piercings were recruited at tattoo conventions, shops (N = 182), and a biannual survey at our STI-outpatient clinic (N = 252) in Amsterdam. Participants were interviewed and tested for anti-HBc and anti-HCV. Determinants of HBV and HCV infections were analysed using logistic regression analysis.

Results

The median number of tattoos and piercings was 5 (IQR 2–10) and 2 (IQR 2–4), respectively. Almost 40% acquired their tattoo of piercing abroad. In total, 18/434 (4.2%, 95%CI: 2.64%–6.46%) participants were anti-HBc positive and 1 was anti-HCV positive (0.2%, 95%CI: 0.01%–1.29%). Being anti-HBc positive was independently associated with older age (OR 1.68, 95%CI: 1.03–2.75 per 10 years older) and being born in an HBV-endemic country (OR 7.39, 95%CI: 2.77–19.7). Tattoo- and/or piercing-related variables, like having a tattoo or piercing in an HBV endemic country, surface percentage tattooed, number of tattoos and piercings etc., were not associated with either HBV or HCV.

Conclusions

We found no evidence for an increased HBV/HCV seroprevalence among persons with multiple tattoos and/or piercings, which might be due to the introduction of hygiene guidelines for tattoo and piercing shops in combination with the low observed prevalence of HBV/HCV in the general population. Tattoos and/or piercings, therefore, should not be considered risk factors for HBV/HCV in the Dutch population. These findings imply the importance of implementation of hygiene guidelines in other countries.     

Binding of oxindole-Schiff base copper(II) complexes to DNA and its modulation by the ligand

Previous studies on copper(II) complexes with oxindole-Schiff base ligands have shown their potential antitumor activity towards different cells, inducing apoptosis through a preferential attack to DNA and/or mitochondria. Herein, we better characterize the interactions between some of these copper(II) complexes and DNA. Investigations on its binding ability to DNA were carried out by fluorescence measurements in competitive experiments with ethidium bromide, using plasmidial or calf-thymus DNA. These results indicated an efficient binding process similar to that observed with copper(II)-phenanthroline species, [Cu(o-phen)₂]²⁺, with binding constants in the range 3 to 9 × 10² M^− 1. DNA cleavage experiments in the presence and absence of distamycin, a recognized binder of DNA, indicated that this binding probably occurs at major or minor groove, leading to double-strand DNA cleavage, and being modulated by the imine ligand. Corroborating these data, discrete changes in EPR spectra of the studied complexes were observed in the presence of DNA, while more remarkable changes were observed in the presence of nucleotides (AMP, GMP, CMP or UMP). Additional evidence for preferential coordination of the copper centers to the bases guanine or cytosine was obtained from titrations of these complexes with each nucleotide, monitored by absorption spectral changes. Therefore, the obtained data point out to their action as groove binders to DNA bases, rather than as intercalators or covalent cross-linkers. Further investigations by SDS PAGE using ³²P-ATP or ³²P-oligonucleotides attested that no hydrolysis of phosphate linkage in DNA or RNA occurs, in the presence of such complexes, confirming their main oxidative mechanism of action.     

MIQuant--semi-automation of infarct size assessment in models of cardiac ischemic injury

Background

The cardiac regenerative potential of newly developed therapies is traditionally evaluated in rodent models of surgically induced myocardial ischemia. A generally accepted key parameter for determining the success of the applied therapy is the infarct size. Although regarded as a gold standard method for infarct size estimation in heart ischemia, histological planimetry is time-consuming and highly variable amongst studies. The purpose of this work is to contribute towards the standardization and simplification of infarct size assessment by providing free access to a novel semi-automated software tool. The acronym MIQuant was attributed to this application.

Methodology/Principal Findings

Mice were subject to permanent coronary artery ligation and the size of chronic infarcts was estimated by area and midline-length methods using manual planimetry and with MIQuant. Repeatability and reproducibility of MIQuant scores were verified. The validation showed high correlation (r ^{midline length} = 0.981; r ^area = 0.970 ) and agreement (Bland-Altman analysis), free from bias for midline length and negligible bias of 1.21% to 3.72% for area quantification. Further analysis demonstrated that MIQuant reduced by 4.5-fold the time spent on the analysis and, importantly, MIQuant effectiveness is independent of user proficiency. The results indicate that MIQuant can be regarded as a better alternative to manual measurement.

Conclusions

We conclude that MIQuant is a reliable and an easy-to-use software for infarct size quantification. The widespread use of MIQuant will contribute towards the standardization of infarct size assessment across studies and, therefore, to the systematization of the evaluation of cardiac regenerative potential of emerging therapies.     

Inhibition of PI3K prevents the proliferation and differentiation of human lung fibroblasts into myofibroblasts: the role of class I P110 isoforms

Idiopathic pulmonary fibrosis (IPF) is a progressive fibroproliferative disease characterized by an accumulation of fibroblasts and myofibroblasts in the alveolar wall. Even though the pathogenesis of this fatal disorder remains unclear, transforming growth factor-β (TGF-β)-induced differentiation and proliferation of myofibroblasts is recognized as a primary event. The molecular pathways involved in TGF-β signalling are generally Smad-dependent yet Smad-independent pathways, including phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt), have been recently proposed. In this research we established ex-vivo cultures of human lung fibroblasts and we investigated the role of the PI3K/Akt pathway in two critical stages of the fibrotic process induced by TGF-β: fibroblast proliferation and differentiation into myofibroblasts. Here we show that the pan-inhibitor of PI3Ks LY294002 is able to abrogate the TGF-β-induced increase in cell proliferation, in α- smooth muscle actin expression and in collagen production besides inhibiting Akt phosphorylation, thus demonstrating the centrality of the PI3K/Akt pathway in lung fibroblast proliferation and differentiation. Moreover, for the first time we show that PI3K p110δ and p110γ are functionally expressed in human lung fibroblasts, in addition to the ubiquitously expressed p110α and β. Finally, results obtained with both selective inhibitors and gene knocking-down experiments demonstrate a major role of p110γ and p110α in both TGF-β-induced fibroblast proliferation and differentiation. This finding suggests that specific PI3K isoforms can be pharmacological targets in IPF.     

Interleukin-6 enhances insulin secretion by increasing glucagon-like peptide-1 secretion from L cells and alpha cells

Exercise, obesity and type 2 diabetes are associated with elevated plasma concentrations of interleukin-6 (IL-6). Glucagon-like peptide-1 (GLP-1) is a hormone that induces insulin secretion. Here we show that administration of IL-6 or elevated IL-6 concentrations in response to exercise stimulate GLP-1 secretion from intestinal L cells and pancreatic alpha cells, improving insulin secretion and glycemia. IL-6 increased GLP-1 production from alpha cells through increased proglucagon (which is encoded by GCG) and prohormone convertase 1/3 expression. In models of type 2 diabetes, the beneficial effects of IL-6 were maintained, and IL-6 neutralization resulted in further elevation of glycemia and reduced pancreatic GLP-1. Hence, IL-6 mediates crosstalk between insulin-sensitive tissues, intestinal L cells and pancreatic islets to adapt to changes in insulin demand. This previously unidentified endocrine loop implicates IL-6 in the regulation of insulin secretion and suggests that drugs modulating this loop may be useful in type 2 diabetes.