Mouse corneal lymphangiogenesis model

This protocol describes a powerful in vivo method to quantitatively study the formation of new lymphatic vessels in the avascular cornea without interference of pre-existing lymphatics. Implantation of 100 ng of lymphangiogenic factors such as vascular endothelial growth factor (VEGF)-A, VEGF-C or fibroblast growth factor-2, together with slow-release polymers, into a surgically created micropocket in the mouse cornea elicits a robust lymphangiogenic response. Newly formed lymphatic vessels are detected by immunohistochemical staining of the flattened corneal tissue with lymphatic endothelial-specific markers such as lymphatic vessel endothelial hyaluronan receptor-1; less-specific markers such as vascular endothelial growth factor receptor 3 may also be used. Lymphatic vessel growth in relation to hemangiogenesis can be readily detected starting at day 5 or 6 after pellet implantation and persists for ～14 d. This protocol offers a unique opportunity to study the mechanisms underlying lymphatic vessel formation, remodeling and function.     

Practical application of self-organizing maps to interrelate biodiversity and functional data in NGS-based metagenomics

Next-generation sequencing (NGS) technologies have enabled the application of broad-scale sequencing in microbial biodiversity and metagenome studies. Biodiversity is usually targeted by classifying 16S ribosomal RNA genes, while metagenomic approaches target metabolic genes. However, both approaches remain isolated, as long as the taxonomic and functional information cannot be interrelated. Techniques like self-organizing maps (SOMs) have been applied to cluster metagenomes into taxon-specific bins in order to link biodiversity with functions, but have not been applied to broad-scale NGS-based metagenomics yet. Here, we provide a novel implementation, demonstrate its potential and practicability, and provide a web-based service for public usage. Evaluation with published data sets mimicking varyingly complex habitats resulted into classification specificities and sensitivities of close to 100% to above 90% from phylum to genus level for assemblies exceeding 8 kb for low and medium complexity data. When applied to five real-world metagenomes of medium complexity from direct pyrosequencing of marine subsurface waters, classifications of assemblies above 2.5 kb were in good agreement with fluorescence in situ hybridizations, indicating that biodiversity was mostly retained within the metagenomes, and confirming high classification specificities. This was validated by two protein-based classifications (PBCs) methods. SOMs were able to retrieve the relevant taxa down to the genus level, while surpassing PBCs in resolution. In order to make the approach accessible to a broad audience, we implemented a feature-rich web-based SOM application named TaxSOM, which is freely available at http://www.megx.net/toolbox/taxsom. TaxSOM can classify reads or assemblies exceeding 2.5 kb with high accuracy and thus assists in linking biodiversity and functions in metagenome studies, which is a precondition to study microbial ecology in a holistic fashion.     

Prediction of BOS by the single-breath nitrogen test in double lung transplant recipients

Background

The present study analyses the ability of the alveolar slope of the single-breath nitrogen washout test (N₂-slope) to diagnose and predict the development of the bronchiolitis obliterans syndrome (BOS).

Methods

We present a retrospective analysis of 61 consecutive bilateral lung or heart-lung transplant recipients who were followed at regular control visits during a three year follow-up. The operating characteristics of the N₂-slope to diagnose BOS and potential BOS (BOS 0-p) and to predict BOS were determined based on cut off values of 95% specificity.

Results

The sensitivity of the N₂-slope to identify BOS was 96%, and BOS 0-p 100%. The predictive ability to predict BOS with a N₂-slope > 478% of the predicted normal was 56%, and if combined with a coincident FEV₁ < 90% of the basal value, the predictive ability was 75%.

Conclusions

The predictive ability of either the N₂-slope or of FEV₁ to diagnose BOS is limited but the combination of the two appears useful. Follow-up protocols of bilateral lung and heart-lung transplant recipients should consider including tests sensitive to obstruction of the peripheral airways.

     

Mycoplasma contamination revisited: mesenchymal stromal cells harboring Mycoplasma hyorhinis potently inhibit lymphocyte proliferation in vitro

Background

Mesenchymal stromal cells (MSC) have important immunomodulatory effects that can be exploited in the clinical setting, e.g. in patients suffering from graft-versus-host disease after allogeneic stem cell transplantation. In an experimental animal model, cultures of rat T lymphocytes were stimulated in vitro either with the mitogen Concanavalin A or with irradiated allogeneic cells in mixed lymphocyte reactions, the latter to simulate allo-immunogenic activation of transplanted T cells in vivo. This study investigated the inhibitory effects of rat bone marrow-derived MSC subsequently found to be infected with a common mycoplasma species (Mycoplasma hyorhinis) on T cell activation in vitro and experimental graft-versus-host disease in vivo.

Principal Findings

We found that M. hyorhinis infection increased the anti-proliferative effect of MSC dramatically, as measured by both radiometric and fluorimetric methods. Inhibition could not be explained solely by the well-known ability of mycoplasmas to degrade tritiated thymidine, but likely was the result of rapid dissemination of M. hyorhinis in the lymphocyte culture.

Conclusions

This study demonstrates the potent inhibitory effect exerted by M. hyorhinis in standard lymphocyte proliferation assays in vitro. MSC are efficient vectors of mycoplasma infection, emphasizing the importance of monitoring cell cultures for contamination.     

Sulindac sulfide reverses aberrant self-renewal of progenitor cells induced by the AML-associated fusion proteins PML/RARα and PLZF/RARα

Chromosomal translocations can lead to the formation of chimeric genes encoding fusion proteins such as PML/RARα, PLZF/RARα, and AML-1/ETO, which are able to induce and maintain acute myeloid leukemia (AML). One key mechanism in leukemogenesis is increased self renewal of leukemic stem cells via aberrant activation of the Wnt signaling pathway. Either X-RAR, PML/RARα and PLZF/RARα or AML-1/ETO activate Wnt signaling by upregulating γ-catenin and β-catenin. In a prospective study, a lower risk of leukemia was observed with aspirin use, which is consistent with numerous studies reporting an inverse association of aspirin with other cancers. Furthermore, a reduction in leukemia risk was associated with use of non-steroidal anti-inflammatory drug (NSAID), where the effects on AML risk was FAB subtype-specific. To better investigate whether NSAID treatment is effective, we used Sulindac Sulfide in X-RARα-positive progenitor cell models. Sulindac Sulfide (SSi) is a derivative of Sulindac, a NSAID known to inactivate Wnt signaling. We found that SSi downregulated both β-catenin and γ-catenin in X-RARα-expressing cells and reversed the leukemic phenotype by reducing stem cell capacity and increasing differentiation potential in X-RARα-positive HSCs. The data presented herein show that SSi inhibits the leukemic cell growth as well as hematopoietic progenitors cells (HPCs) expressing PML/RARα, and it indicates that Sulindac is a valid molecular therapeutic approach that should be further validated using in vivo leukemia models and in clinical settings.     

Prostate cancer cell lines under hypoxia exhibit greater stem-like properties

Hypoxia is an important environmental change in many cancers. Hypoxic niches can be occupied by cancer stem/progenitor-like cells that are associated with tumor progression and resistance to radiotherapy and chemotherapy. However, it has not yet been fully elucidated how hypoxia influences the stem-like properties of prostate cancer cells. In this report, we investigated the effects of hypoxia on human prostate cancer cell lines, PC-3 and DU145. In comparison to normoxia (20% O(2)), 7% O(2) induced higher expressions of HIF-1α and HIF-2α, which were associated with upregulation of Oct3/4 and Nanog; 1% O(2) induced even greater levels of these factors. The upregulated NANOG mRNA expression in hypoxia was confirmed to be predominantly retrogene NANOGP8. Similar growth rates were observed for cells cultivated under hypoxic and normoxic conditions for 48 hours; however, the colony formation assay revealed that 48 hours of hypoxic pretreatment resulted in the formation of more colonies. Treatment with 1% O(2) also extended the G(0)/G(1) stage, resulting in more side population cells, and induced CD44 and ABCG2 expressions. Hypoxia also increased the number of cells positive for ABCG2 expression, which were predominantly found to be CD44(bright) cells. Correspondingly, the sorted CD44(bright) cells expressed higher levels of ABCG2, Oct3/4, and Nanog than CD44(dim) cells, and hypoxic pretreatment significantly increased the expressions of these factors. CD44(bright) cells under normoxia formed significantly more colonies and spheres compared with the CD44(dim) cells, and hypoxic pretreatment even increased this effect. Our data indicate that prostate cancer cells under hypoxia possess greater stem-like properties.     

Genome-wide miRNA signatures of human longevity

Little is known about the functions of miRNAs in human longevity. Here, we present the first genome-wide miRNA study in long-lived individuals (LLI) who are considered a model for healthy aging. Using a microarray with 863 miRNAs, we compared the expression profiles obtained from blood samples of 15 centenarians and nonagenarians (mean age 96.4 years) with those of 55 younger individuals (mean age 45.9 years). Eighty miRNAs showed aging-associated expression changes, with 16 miRNAs being up-regulated and 64 down-regulated in the LLI relative to the younger probands. Seven of the eight selected aging-related biomarkers were technically validated using quantitative RT-PCR, confirming the microarray data. Three of the eight miRNAs were further investigated in independent samples of 15 LLI and 17 younger participants (mean age 101.5 and 36.9 years, respectively). Our screening confirmed previously published miRNAs of human aging, thus reflecting the utility of the applied approach. The hierarchical clustering analysis of the miRNA microarray expression data revealed a distinct separation between the LLI and the younger controls (P-value < 10(-5) ). The down-regulated miRNAs appeared as a cluster and were more often reported in the context of diseases than the up-regulated miRNAs. Moreover, many of the differentially regulated miRNAs are known to exhibit contrasting expression patterns in major age-related diseases. Further in silico analyses showed enrichment of potential targets of the down-regulated miRNAs in p53 and other cancer pathways. Altogether, synchronized miRNA-p53 activities could be involved in the prevention of tumorigenesis and the maintenance of genomic integrity during aging.     

Genome wide adaptations of Plasmodium falciparum in response to lumefantrine selective drug pressure

The combination therapy of the Artemisinin-derivative Artemether (ART) with Lumefantrine (LM) (Coartem®) is an important malaria treatment regimen in many endemic countries. Resistance to Artemisinin has already been reported, and it is feared that LM resistance (LMR) could also evolve quickly. Therefore molecular markers which can be used to track Coartem® efficacy are urgently needed. Often, stable resistance arises from initial, unstable phenotypes that can be identified in vitro. Here we have used the Plasmodium falciparum multidrug resistant reference strain V1S to induce LMR in vitro by culturing the parasite under continuous drug pressure for 16 months. The initial IC₅₀ (inhibitory concentration that kills 50% of the parasite population) was 24 nM. The resulting resistant strain V1S_LM, obtained after culture for an estimated 166 cycles under LM pressure, grew steadily in 378 nM of LM, corresponding to 15 times the IC₅₀ of the parental strain. However, after two weeks of culturing V1S_LM in drug-free medium, the IC₅₀ returned to that of the initial, parental strain V1S. This transient drug tolerance was associated with major changes in gene expression profiles: using the PFSANGER Affymetrix custom array, we identified 184 differentially expressed genes in V1S_LM. Among those are 18 known and putative transporters including the multidrug resistance gene 1 (pfmdr1), the multidrug resistance associated protein and the V-type H+ pumping pyrophosphatase 2 (pfvp2) as well as genes associated with fatty acid metabolism. In addition we detected a clear selective advantage provided by two genomic loci in parasites grown under LM drug pressure, suggesting that all, or some of those genes contribute to development of LM tolerance – they may prove useful as molecular markers to monitor P. falciparum LM susceptibility.     

Multilocus sequence typing of genital Chlamydia trachomatis in Norway reveals multiple new sequence types and a large genetic diversity

Background

The Chlamydia trachomatis incidence rate in Finnmark, the most northern and sparsely populated county in Norway, has been twice the national average. This population based cross-sectional study among Finnmark high school students had the following aims: i) to examine distribution of multilocus sequence types (STs) of C. trachomatis in a previously unmapped area, ii) to compare chlamydia genetic diversity in Finnmark with that of two urban regions, and iii) to compare discriminatory capacity of multilocus sequence typing (MLST) with conventional ompA sequencing in a large number of chlamydia specimens.

Methodology

ompA sequencing and a high-resolution MLST system based on PCR amplification and DNA sequencing of five highly variable genetic regions were used. Eighty chlamydia specimens from adolescents aged 15–20 years in Finnmark were collected in five high schools (n = 60) and from routine clinical samples in the laboratory (n = 20). These were compared to routine clinical samples from adolescents in Tromsø (n = 80) and Trondheim (n = 88), capitals of North and Central Norway, respectively.

Principal Findings

ompA sequencing detected 11 genotypes in 248 specimens from all three areas. MLST displayed 50 STs providing a five-fold higher resolution. Two-thirds of all STs were novel. The common ompA E/Bour genotype comprised 46% and resolved into 24 different STs. MLST identified the Swedish new variant of C. trachomatis not discriminated by ompA sequencing. Simpson''s discriminatory index (D) was 0.93 for MLST, while a corrected D_c was 0.97. There were no statistically significant differences in ST genetic diversity between geographic areas. Finnmark had an atypical genovar distribution with G being predominant. This was mainly due to expansion of specific STs of which the novel ST161 was unique for Finnmark.

Conclusions/Significance

MLST revealed multiple new STs and a larger genetic diversity in comparison to ompA sequencing and proved to be a useful tool in molecular epidemiology of chlamydia infections.