The Relative Composition of the Inflammatory Infiltrate as an Additional Tool for Synovial Tissue Classification

Objectives

Traditionally, differences in absolute numbers of cells expressing a certain marker (e.g., positive staining cells per mm²) have been used in immunohistological synovial tissue classification. We have begun to evaluate the relative composition of the inflammatory infiltrates, i.e. percentages of inflammatory cell types in inflammatory infiltrates, as an alternate classification tool that may potentially improve tissue diagnostics, subgrouping in clinical trials, and understanding of pathogenesis of inflammatory and noninflammatory arthropathies.

Methods

Synovial tissue specimens (normal synovium, n=15; orthopedic arthropathies, n=6; osteoarthritis, n=26; early undifferentiated arthritis, n=10; rheumatoid arthritis, n=26; chronic septic arthritis, n=11) were stained for CD15, CD68, CD3, CD20, and CD38. Densities of cells expressing a given marker were determined in the superficial subintima. Binary and multicategory receiver operating characteristic (ROC) analysis and naïve Bayes classifier were used to compare the abilities of (1) the absolute densities of cells expressing a given marker (absolute method) with (2) the percentages of these cells in the inflammatory cell population (relative method) to differentiate among the six tissue classes.

Results

The inflammatory infiltrates in normal synovium and the orthopedic arthropathies consisted almost exclusively of CD68+ and CD3+ cells. Notable fractions of CD20+ and CD38+ cells appeared in a subset of osteoarthritis samples, and increased further in early, rheumatoid and chronic septic arthritis. ROC analyses and naïve Bayes classifier ranked the absolute method above the relative method in terms of overall discriminatory ability. The relative method became slightly superior when the samples were also stratified according to the total number of inflammatory cells/mm².

Conclusions

This exploratory investigation featuring a variety of joint disorders revealed that measuring the relative proportions of inflammatory cell types may aid in synovial tissue classification if the samples are also stratified according to the intensity of inflammation.     

Multiple improvement of astaxanthin biosynthesis in Xanthophyllomyces dendrorhous by a combination of conventional mutagenesis and metabolic pathway engineering

Xanthophyllomyces dendrorhous (Phaffia rhodozyma) is the only yeast or fungus that synthesizes the commercially attractive carotenoid astaxanthin. For a suitable bioprocess, the wild type has to be modified for increasing biomass content. To achieve this, a two step strategy has been followed. At first, random mutagenesis was applied leading to colonies with substantially higher astaxanthin content. Then, the resulting strain was genetically engineered by targeting limiting reactions for further enhancement of astaxanthin biosynthesis. This combinatorial approach together with selection of an appropriate growth medium resulted in highest astaxanthin biomass contents reported to date for X. dendrorhous. In a fermenter culture, its maximum content was 9.7 mg/g dry weight.     

Nep1-like proteins as a target for plant pathogen control

The lack of efficient methods to control the major diseases of crops most important to agriculture leads to huge economic losses and seriously threatens global food security. Many of the most important microbial plant pathogens, including bacteria, fungi, and oomycetes, secrete necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs), which critically contribute to the virulence and spread of the disease. NLPs are cytotoxic to eudicot plants, as they disturb the plant plasma membrane by binding to specific plant membrane sphingolipid receptors. Their pivotal role in plant infection and broad taxonomic distribution makes NLPs a promising target for the development of novel phytopharmaceutical compounds. To identify compounds that bind to NLPs from the oomycetes Pythium aphanidermatum and Phytophthora parasitica, a library of 587 small molecules, most of which are commercially unavailable, was screened by surface plasmon resonance. Importantly, compounds that exhibited the highest affinity to NLPs were also found to inhibit NLP-mediated necrosis in tobacco leaves and Phytophthora infestans growth on potato leaves. Saturation transfer difference-nuclear magnetic resonance and molecular modelling of the most promising compound, anthranilic acid derivative, confirmed stable binding to the NLP protein, which resulted in decreased necrotic activity and reduced ion leakage from tobacco leaves. We, therefore, confirmed that NLPs are an appealing target for the development of novel phytopharmaceutical agents and strategies, which aim to directly interfere with the function of these major microbial virulence factors. The compounds identified in this study represent lead structures for further optimization and antimicrobial product development.     

Early setting of grammatical processing in the bilingual brain

The existence of a "critical period" for language acquisition is controversial. Bilingual subjects with variable age of acquisition (AOA) and proficiency level (PL) constitute a suitable model to study this issue. We used functional magnetic resonance imaging to investigate the effects of AOA and PL on neural correlates of grammatical and semantic judgments in Italian-German bilinguals who learned the second language at different ages and had different proficiency levels. While the pattern of brain activity for semantic judgment was largely dependent on PL, AOA mainly affected the cortical representation of grammatical processes. These findings support the view that both AOA and PL affect the neural substrates of second language processing, with a differential effect on grammar and semantics.     

A Comparative Study of Germination Biology and Plant Performance in Two Dry Grassland Species

Over the last 20 years, the species assemblage of the species-rich dry grassland communities of central Germany has changed due to the ongoing abandonment of traditional land-use practices. In our study we wanted to investigate the germination biology and the plant-plant interaction of the low-growing and declining forb Alyssum montanum and the increasingly dominant grass Festuca rupicola. To investigate the germination behaviour we simulated cold (8/4°C), warm (20/10°C) and hot (32/20°C) conditions under a light-dark regime (12/12 hours) as well as in constant darkness. Germination of both species was similar with almost all non-dormant seeds germinating under intermediate temperature conditions (20/10°C). Whereas F. rupicola (F) germinated equally well under light changing conditions and constant darkness, the germination of A. montanum (A) was clearly reduced in darkness, which implies a strong competitive advantage of F. rupicola in dense vegetation cover. However, A. montanum germinated early under all temperatures regimes and better under 20/10°C as well as with light changing conditions. The interactions between the two species were tested in a pod-experiment with a replacement design, within which the composition of individuals was changed in the following way: 4A/1F, 3A/2F, 2A/3F, 1A/4F (eight replications per each). To evaluate any influence of intraspecific competition we created monocultures in an additive design (1A, 2A, 3A, 4A, 5A, 1F, 5F; eight replications per each). Despite that A. montanum developed faster and flowered after only four months in the replacement design experiment, the herb responded negatively to the presence of F. rupicola in several parameters (number of flowers, number of fruits and biomass per individual). The calculation of the Relative Neighbour Effect (RNE) indicated facilitation for F. rupicola and competition on A. montanum, which increased with increasing number of individuals of the other species. Our data suggest that A. montanum seedling recruitment, growth and fitness are negatively affected by increasing vegetation density of the expanding grass species F. rupicola.     

Prevailing negative soil biota effect and no evidence for local adaptation in a widespread Eurasian grass

Background

Soil biota effects are increasingly accepted as an important driver of the abundance and distribution of plants. While biogeographical studies on alien invasive plant species have indicated coevolution with soil biota in their native distribution range, it is unknown whether adaptation to soil biota varies among populations within the native distribution range. The question of local adaptation between plants and their soil biota has important implications for conservation of biodiversity and may justify the use of seed material from local provenances in restoration campaigns.

Methodology/Principal Findings

We studied soil biota effects in ten populations of the steppe grass Stipa capillata from two distinct regions, Europe and Asia. We tested for local adaptation at two different scales, both within (ca. 10–80 km) and between (ca. 3300 km) regions, using a reciprocal inoculation experiment in the greenhouse for nine months. Generally, negative soil biota effects were consistent. However, we did not find evidence for local adaptation: both within and between regions, growth of plants in their ‘home soil’ was not significantly larger relative to that in soil from other, more distant, populations.

Conclusions/Significance

Our study suggests that negative soil biota effects can prevail in different parts of a plant species'' range. Absence of local adaptation points to the possibility of similar rhizosphere biota composition across populations and regions, sufficient gene flow to prevent coevolution, selection in favor of plasticity, or functional redundancy among different soil biota. From the point of view of plant - soil biota interactions, our findings indicate that the current practice of using seeds exclusively from local provenances in ecosystem restoration campaigns may not be justified.     

Historic and recent fragmentation coupled with altitude affect the genetic population structure of one of the world's highest tropical tree line species

Aim To assess the effects of altitude and historic and recent forest fragmentation on the genetic diversity and structure of the wind‐pollinated tropical tree line species Polylepis incana. Location One of the highest mountain forest regions of the world, located in the Eastern Cordillera of the Ecuadorian Andes. Methods We compared genetic diversity and structure of adult trees with those of seedlings (n= 118 in both cases) in nine forest stands spanning an altitudinal gradient from 3500 to 4100 m a.s.l. using amplified fragment length polymorphisms (AFLPs). Genetic diversity was calculated as percentage of polymorphic bands (P) and Nei's expected heterozygosity (He); genetic differentiation was assessed using analysis of molecular variance, Φ_ST statistics and Bayesian cluster analysis. Results Estimates of genetic diversity at the population level were significantly lower in seedlings than in adults. Genetic diversity (He‐value) was, in both cases, negatively correlated to altitude and positively correlated to population size in the seedlings. Genetic differentiation of the seedlings was approximately as high (φ_ST= 0.298) as that of the adults (φ_ST= 0.307), and geographical differentiation was clearly reflected in both AFLP profiles, with mountain ridges acting as barriers to gene flow. Main conclusions Our study provides evidence of a historic upslope migration of P. incana in central Ecuador. In addition, it highlights the detrimental effects of unexpectedly strong genetic isolation, both recent and historical, particularly for our wind‐pollinated species where the distance between forest stands was less than 25 km. We therefore additionally propose that in habitats with pronounced high‐mountain landscape structures, gene flow may be hampered to such an extent that species have a more pronounced sensitivity to habitat fragmentation, even among populations of wind‐pollinated trees.     

Mitoxantrone induces natural killer cell maturation in patients with secondary progressive multiple sclerosis

Mitoxantrone is one of the few drugs approved for the treatment of progressive multiple sclerosis (MS). However, the prolonged use of this potent immunosuppressive agent is limited by the appearance of severe side effects. Apart from its general cytotoxic effect, the mode of action of mitoxantrone on the immune system is poorly understood. Thus, to develop safe therapeutic approaches for patients with progressive MS, it is essential to elucidate how mitoxantrone exerts it benefits. Accordingly, we initiated a prospective single-arm open-label study with 19 secondary progressive MS patients. We investigated long-term effects of mitoxantrone on patient peripheral immune subsets using flow cytometry. While we corroborate that mitoxantrone persistently suppresses B cells in vivo, we show for the first time that treatment led to an enrichment of neutrophils and immunomodulatory CD8(low) T cells. Moreover, sustained mitoxantrone applications promoted not only persistent NK cell enrichment but also NK cell maturation. Importantly, this mitoxantrone-induced NK cell maturation was seen only in patients that showed a clinical response to treatment. Our data emphasize the complex immunomodulatory role of mitoxantrone, which may account for its benefit in MS. In particular, these results highlight the contribution of NK cells to mitoxantrone efficacy in progressive MS.     

Transposon-mediated BAC transgenesis in human ES cells

Transgenesis is a cornerstone of molecular biology. The ability to integrate a specifically engineered piece of DNA into the genome of a living system is fundamental to our efforts to understand life and exploit its implications for medicine, nanotechnology and bioprospecting. However, transgenesis has been hampered by position effects and multi-copy integration problems, which are mainly due to the use of small, plasmid-based transgenes. Large transgenes based on native genomic regions cloned into bacterial artificial chromosomes (BACs) circumvent these problems but are prone to fragmentation. Herein, we report that contrary to widely held notions, large BAC-sized constructs do not prohibit transposition. We also report the first reliable method for BAC transgenesis in human embryonic stem cells (hESCs). The PiggyBac or Sleeping Beauty transposon inverted repeats were integrated into BAC vectors by recombineering, followed by co-lipofection with the corresponding transposase in hESCs to generate robust fluorescent protein reporter lines for OCT4, NANOG, GATA4 and PAX6. BAC transposition delivers several advantages, including increased frequencies of single-copy, full-length integration, which will be useful in all transgenic systems but especially in difficult venues like hESCs.