Persistence of Epstein-Barr Virus in Self-Reactive Memory B Cells

Epstein-Barr virus infection has been epidemiologically associated with the development of multiple autoimmune diseases, particularly systemic lupus erythematosus and multiple sclerosis. Currently, there is no known mechanism that can account for these associations. The germinal-center (GC) model of EBV infection and persistence proposes that EBV gains access to the memory B cell compartment via GC reactions by driving infected cells to differentiate using the virus-encoded LMP1 and LMP2a proteins, which act as functional homologues of CD40 and the B cell receptor, respectively. The ability of LMP2a, when expressed in mice, to allow escape of autoreactive B cells suggests that it could perform a similar role in infected GC B cells, permitting the survival of potentially pathogenic autoreactive B cells. To test this hypothesis, we cloned and expressed antibodies from EBV⁺ and EBV⁻ memory B cells present during acute infection and profiled their self- and polyreactivity. We find that EBV does persist within self- and polyreactive B cells but find no evidence that it favors the survival of pathogenic autoreactive B cells. On the contrary, EBV⁺ memory B cells express lower levels of self-reactive and especially polyreactive antibodies than their uninfected counterparts do. Our work suggests that EBV has only a modest effect on the GC process, which allows it to access and persist within a subtly unique niche of the memory compartment characterized by relatively low levels of self- and polyreactivity. We suggest that this might reflect an active process where EBV and its human host have coevolved so as to minimize the virus''s potential to contribute to autoimmune disease.     

Left Ventricular Dysfunction and CXCR3 Ligands in Hypertension: From Animal Experiments to a Population-Based Pilot Study

Detecting left ventricular (LV) dysfunction at an early stage is key in addressing the heart failure epidemic. In proteome profiling experiments in mice subjected either to aortic banding or sham, the circulating CXCR3 ligands monokine induced by interferon-γ (MIG) and interferon-γ inducible protein 10 (IP10) were 5 to 40 fold up-regulated at eight weeks. We assessed the diagnostic value of circulating NT-pro BNP and CXCR3 ligands (MIG, IP10, Interferon-inducible T-cell alpha chemo-attractant [I–TAC]) in patients with hypertension (≥140/90 mm Hg) associated with subclinical (n = 19) or symptomatic (n = 16) diastolic LV dysfunction on echocardiography and healthy controls. NT–pro BNP, MIG, IP10, I–TAC all increased (p ≤ 0.014) across the categories of worsening left ventricular dysfunction. In patients with symptomatic disease, MIG, IP10, and I–TAC increased 210% (p = 0.015), 140% (p = 0.007) and 120% (p = 0.035) more than NT-pro BNP. The optimal discrimination limits, obtained by maximizing Youden’s index were 246 pmol/L, 65 pg/mL, 93 pg/mL, and 24 pg/mL, respectively. The odds ratios associated with the four biomarkers were significant (p ≤ 0.010), ranging from 4.00 for IP10 to 9.69 for MIG. With adjustment for NT–pro BNP, the CXCR3 ligands retained significance (p ≤ 0.028). Adding optimized thresholds for the CXCR3 ligands to NT–pro BNP enhanced (p ≤ 0.014) the integrated discrimination improvement and the net reclassification improvement. In conclusion, congruent with the concept that inflammation plays a key role in the pathogenesis of LV dysfunction, MIG, IP10 and I–TAC add diagnostic accuracy over and beyond NT–pro BNP.     

Affinity of integrin alpha 1 beta 1 from liver sinusoidal membranes for type IV collagen.

Hepatic sinusoidal membranes isolated from adult rats were extracted with detergent and fractionated on a wheat germ agglutinin affinity column. Bound glycoproteins were eluted with N-acetyl glucosamine and chromatographed on a type IV collagen affinity column. Recovery of the bound fraction by EDTA and analysis by SDS-PAGE revealed two glycoproteins with apparent molecular weights of 180,000 and 117,000. These were identified immunologically by Western blotting as the alpha and beta subunits of integrin alpha 1 beta 1.     

Isolation and characterization of a glycosylated form of human insulin-like growth factor I produced in Saccharomyces cerevisiae

Expression and secretion of human insulin-like growth factor-I (IGF-I) in Saccharomyces cerevisiae was achieved by linking an actin (ACT) promoter to an MF alpha 1 prepro leader peptide/IGF-I gene fusion. Purified human IGF-I from yeast culture media was found to contain, in addition to the native form, also a glycosylated variant. Structural studies showed that both IGF-I forms were processed identically, resulting in 70-amino-acid long polypeptides, with intact N-terminal and C-terminal residues of glycine and alanine, respectively. The glycosylation site was determined to threonine-29 (Thr29), by 1H NMR spectroscopy and protein sequence analysis of an isolated tryptic peptide(22-36). No other glycosylation sites were found. Only mannose was detected in the sugar analysis, with an estimated content of 4.5% w/w corresponding to 2 mannose residues per molecule of IGF-I. The carbohydrate structure, determined by 1H and 13C NMR spectroscopy, was found to be alpha-D-Manp(1----2)alpha-D-Manp(1----3)Thr corresponding to an O-linked glycoprotein structure. No other post-translational modifications could be identified in the glycosylated IGF-I form. Furthermore, this form was highly active, comparable to native IGF-I, exhibiting a specific activity of 20,500 units/mg, as determined by a radio-receptor assay.     

Regeneration of a Gentiana pneumonanthe population in an oligotrophic wet meadow

Abstract. The aim of this study was to evaluate the critical phases of the life cycle of Gentiana pneumonanthe, the marsh gentian, a threatened species of the Bohemian flora. The effect of various conditions on germination and seedling establishment and the possible effect of competition on the performance of the species were tested. Seeds were sown in plots which were subjected to four treatments in a randomized complete blocks experiment: unmanaged meadow, mown meadow, burned meadow, and meadow with cut sod. The significantly highest recruitment was found in plots with cut sod, the lowest in the unmanaged control plots. Seedling survival also differed among the treatments. In the following year surviving individuals were only found in plots with cut sod. The influence of neighbouring vegetation on target gentian individuals was evaluated by removing the surrounding vegetation and comparing the performance of these individuals with controls. The initial height of each individual was measured and used as a covariable. No significant effect of neighbouring vegetation on performance was detected. Thus, the establishment phase appears to be critical for population persistence and is also more influenced by the management regime than other stages of the life cycle.     

A simulation approach for power calculation in large cohort studies based on multistate models

Realistic power calculations for large cohort studies and nested case control studies are essential for successfully answering important and complex research questions in epidemiology and clinical medicine. For this, we provide a methodical framework for general realistic power calculations via simulations that we put into practice by means of an R‐based template. We consider staggered recruitment and individual hazard rates, competing risks, interaction effects, and the misclassification of covariates. The study cohort is assembled with respect to given age‐, gender‐, and community distributions. Nested case‐control analyses with a varying number of controls enable comparisons of power with a full cohort analysis. Time‐to‐event generation under competing risks, including delayed study‐entry times, is realized on the basis of a six‐state Markov model. Incidence rates, prevalence of risk factors and prefixed hazard ratios allow for the assignment of age‐dependent transition rates given in the form of Cox models. These provide the basis for a central simulation‐algorithm, which is used for the generation of sample paths of the underlying time‐inhomogeneous Markov processes. With the inclusion of frailty terms into the Cox models the Markov property is specifically biased. An “individual Markov process given frailty” creates some unobserved heterogeneity between individuals. Different left‐truncation‐ and right‐censoring patterns call for the use of Cox models for data analysis. p‐values are recorded over repeated simulation runs to allow for the desired power calculations. For illustration, we consider scenarios with a “testing” character as well as realistic scenarios. This enables the validation of a correct implementation of theoretical concepts and concrete sample size recommendations against an actual epidemiological background, here given with possible substudy designs within the German National Cohort.