Possible mechanisms of allergy prevention: dendritic cells as regulators of the allergy-protective immune response induced by cowshed bacteria

Aktuelle epidemiologische Studien belegen, dass Kinder, die in einer bäuerlichen Umgebung aufwachsen, eine geringere Inzidenz für atopische Sensibilisierung, Heuschnupfen und Asthma aufweisen, als Kinder im selben Alter, die nicht in dieser Umgebung leben. Diese Befunde unterstützen die Hygiene-Hypothese, welche besagt, dass ein Kontakt zu einer Mikroorganismen-reichen Umgebung in den ersten Lebensjahren die Entwicklung von allergischen Reaktionen im späteren Leben beeinflusst. Da die ersten Lebensjahre wichtig für die Reifung des Immunsystems und der Entwicklung einer gesunden THelfer (TH)-Zell-Balance (TH1/TH2/TReg) sind, könnten die mikrobiellen Komponenten eine entscheidende Rolle bei der Ausrichtung diese Balance spielen, indem sie entweder TH1-Antworten begünstigen und dadurch die allergieassoziierte TH2-Antwort unterdrücken oder indem sie TReg-Antworten induzieren und damit generell alle überschießenden Immunantworten supprimieren. Daraus entstand die Frage, ob Mikroorgansimen der bäuerlichen Umgebung und/oder ihre Produkte in der Lage sind, die Immunantworten in einer dieser Weisen zu modulieren. Um die potentielle allergieprotektive Wirkung dieser Mikroorgansimen zu untersuchen, wurden zwei aus Kuhställen isolierte Bakterienstämme, Acinetobacter lwoffii F78 (A. lwoffii F78) und Lactococcus lactis G121 (L. lactis G121), ausgewählt und charakterisiert. Tatsächlich konnte in einem Mausmodell für allergisches Asthma gezeigt werden, dass beide Kuhstallisolate die allergischen Reaktionen der Mäuse reprimieren. Da unklar blieb, welche bakteriellen Moleküle und welche Rezeptoren und nachfolgenden Signaltransduktionswege beteiligt waren, wurde die Fähigkeit der Bakterien Immunantworten zu modulieren in humanen dendritischen Zellen (DCs) untersucht. Beide Bakterienstämme induzierten die Aktivierung und Reifung der DCs und führten zu einer stark TH1-polarisierten Immunantwort. Zudem konnten keine Anzeichen für die Induktion einer TH2- oder TReg- Antwort nachgewiesen werden. Um die für die TH1-Polarisierung verantwortlichen Strukturen der Bakterien näher zu charakterisieren wurde die Beteiligung verschiedener Rezeptoren des angeborenen Immunsystems untersucht. Diese Studien zeigten, dass der TLR4-Ligand von A. lwoffii F78, das LPS der äußeren Membran, für die TH1-polarisierenden Effekte verantwortlich ist und zumindest für die allergieprotektiven Fähigkeiten die bedeutendste Struktur dieses Bakterienstammes ist. Im Hinblick auf L. lactis G121 konnte die Notwendigkeit der zellulären Aufnahme des Bakteriums und damit die Involvierung eines intrazellulären Rezeptors, wahrscheinlich NOD2, für die TH1-polarisierenden Effekte nachgewiesen werden. Zudem induzierte L. lactis G121 eine untypische Zellkerntranslokation der NF-κB-Untereinheit c-Rel ohne gleichzeitige Translokation der p65- Untereinheit. Weiterhin konnte eine bis zu 48 h anhaltende Aktivierung der DCs durch L. lactis G121 festgestellt werden. Dies könnte ein wichtiger Mechanismus für eine dauerhafte Induktion einer anti-allergischen Immunantwort sein. Insgesamt untermauern diese Daten die Hygiene-Hypothese und implizieren dass die TH1- polarisierenden Eigenschaften der Bakterien wichtige Mechanismen für die allergieprotektiven Effekte der bäuerlichen Umgebung darstellen. Zudem liefert diese Arbeit erste Ansatzpunkte für die Entwicklung von allergieprotektiven Therapien mit einer potentiellen Anwendung der Kuhstallbakterien.Recent epidemiological studies clearly indicate that children who grow up in a farming environment show lower levels of atopic sensitization, hay fever and asthma than children of the same age living not in such an environment. These findings support the hygiene hypothesis, stating that an early-life contact to an environment rich in microbial compounds influences the development of allergic reactions later in life. As the first year of life is important for the maturation of the immune system and the establishment of the THelper (TH) cell balance (TH1/TH2/TReg) such microbial contact might play a pivotal role in adjusting this balance by either promoting TH1 responses and thereby inhibiting allergy-associated TH2 responses or promoting TRegs and thereby suppress in general overwhelming immune responses. Therefore the question arose if farm microbial organisms and/or their products may have the ability to modulate immune responses in either way. To gain further insight into the potential allergy-protective properties of farm microbes two bacterial species isolated from cowsheds of farms were selected and characterized, namely Acinetobacter lwoffii F78 (A. lwoffii F78) and Lactococcus lactis G121 (L. lactis G121). In fact, it could be shown that both bacterial isolates were able to reduce allergic reactions in a mouse model of allergic asthma. Since it remained unclear which bacterial molecules and which receptors and subsequent signal transduction pathways were involved, the immune modulatory capacities of the bacteria with focus on their influence on the TH cell balance were examined in human dendritic cells (DCs) as these cells provide a number of essential signals for T cell priming. Both bacterial strains induced activation and maturation of DCs and led to a strong TH1- polarizing immune response. Furthermore, no evidence for the induction of TH2 or TReg cells could be detected. To further characterize the microbial structures responsible for the allergyprotective properties the involvement of innate immune receptors that participated in recognition of the two bacteria strains was analyzed. These studies revealed that the TLR4 ligand from A. lwoffii F78, the LPS of the outer membrane is responsible for the TH1- polarizing effects of A. lwoffii F78 and, at least with respect to the allergy-protective properties, seems to be the most dominant structure of this strain. With respect to L. lactis G121, the requirement of phagocytic uptake and thus an intracellular receptor, which most likely is NOD2, could be shown for the TH1-polarizing effects. In addition, L. lactis G121 induced an atypical nuclear translocation of the NF-κB subunit c-Rel without translocation of the p65 subunit. Moreover, a sustained activation of DCs for up to 48 h was detected. This might be an important mechanism for a prolonged triggering of an anti-allergic immune response. Together, these data strongly support the hygiene hypothesis and imply that the TH1- polarization properties of the bacteria are important mechanisms for the allergy-protective farm effects. In addition, this work provides first starting points for the development of allergyprotective therapies with potential application of the cowshed bacteria

Untersuchungen zur Interaktion von Ipomoea batatas mit synergistischen und pathogenen Mikroorganismen

[no abstract

Discovery and assembly of repeat family pseudomolecules from sparse genomic sequence data using the Assisted Automated Assembler of Repeat Families (AAARF) algorithm

<p>Abstract</p> <p>Background</p> <p>Higher eukaryotic genomes are typically large, complex and filled with both genes and multiple classes of repetitive DNA. The repetitive DNAs, primarily transposable elements, are a rapidly evolving genome component that can provide the raw material for novel selected functions and also indicate the mechanisms and history of genome evolution in any ancestral lineage. Despite their abundance, universality and significance, studies of genomic repeat content have been largely limited to analyses of the repeats in fully sequenced genomes.</p> <p>Results</p> <p>In order to facilitate a broader range of repeat analyses, the Assisted Automated Assembler of Repeat Families algorithm has been developed. This program, written in PERL and with numerous adjustable parameters, identifies sequence overlaps in small shotgun sequence datasets and walks them out to create long pseudomolecules representing the most abundant repeats in any genome. Testing of this program in maize indicated that it found and assembled all of the major repeats in one or more pseudomolecules, including coverage of the major Long Terminal Repeat retrotransposon families. Both Sanger sequence and 454 datasets were appropriate.</p> <p>Conclusion</p> <p>These results now indicate that hundreds of higher eukaryotic genomes can be efficiently characterized for the nature, abundance and evolution of their major repetitive DNA components.</p

MaHPIC malaria systems biology data from Plasmodium cynomolgi sporozoite longitudinal infections in macaques

Plasmodium cynomolgi causes zoonotic malarial infections in Southeast Asia and this parasite species is important as a model for Plasmodium vivax and Plasmodium ovale. Each of these species produces hypnozoites in the liver, which can cause relapsing infections in the blood. Here we present methods and data generated from iterative longitudinal systems biology infection experiments designed and performed by the Malaria Host-Pathogen Interaction Center (MaHPIC) to delve deeper into the biology, pathogenesis, and immune responses of P. cynomolgi in the Macaca mulatta host. Infections were initiated by sporozoite inoculation. Blood and bone marrow samples were collected at defined timepoints for biological and computational experiments and integrative analyses revolving around primary illness, relapse illness, and subsequent disease and immune response patterns. Parasitological, clinical, haematological, immune response, and -omic datasets (transcriptomics, proteomics, metabolomics, and lipidomics) including metadata and computational results have been deposited in public repositories. The scope and depth of these datasets are unprecedented in studies of malaria, and they are projected to be a F.A.I.R., reliable data resource for decades

PacBio assembly of a Plasmodium knowlesi genome sequence with Hi-C correction and manual annotation of the SICAvar gene family.

Plasmodium knowlesi has risen in importance as a zoonotic parasite that has been causing regular episodes of malaria throughout South East Asia. The P. knowlesi genome sequence generated in 2008 highlighted and confirmed many similarities and differences in Plasmodium species, including a global view of several multigene families, such as the large SICAvar multigene family encoding the variant antigens known as the schizont-infected cell agglutination proteins. However, repetitive DNA sequences are the bane of any genome project, and this and other Plasmodium genome projects have not been immune to the gaps, rearrangements and other pitfalls created by these genomic features. Today, long-read PacBio and chromatin conformation technologies are overcoming such obstacles. Here, based on the use of these technologies, we present a highly refined de novo P. knowlesi genome sequence of the Pk1(A+) clone. This sequence and annotation, referred to as the 'MaHPIC Pk genome sequence', includes manual annotation of the SICAvar gene family with 136 full-length members categorized as type I or II. This sequence provides a framework that will permit a better understanding of the SICAvar repertoire, selective pressures acting on this gene family and mechanisms of antigenic variation in this species and other pathogens

A New Single-Step PCR Assay for the Detection of the Zoonotic Malaria Parasite Plasmodium knowlesi

Recent studies in Southeast Asia have demonstrated substantial zoonotic transmission of Plasmodium knowlesi to humans. Microscopically, P. knowlesi exhibits several stage-dependent morphological similarities to P. malariae and P. falciparum. These similarities often lead to misdiagnosis of P. knowlesi as either P. malariae or P. falciparum and PCR-based molecular diagnostic tests are required to accurately detect P. knowlesi in humans. The most commonly used PCR test has been found to give false positive results, especially with a proportion of P. vivax isolates. To address the need for more sensitive and specific diagnostic tests for the accurate diagnosis of P. knowlesi, we report development of a new single-step PCR assay that uses novel genomic targets to accurately detect this infection.We have developed a bioinformatics approach to search the available malaria parasite genome database for the identification of suitable DNA sequences relevant for molecular diagnostic tests. Using this approach, we have identified multi-copy DNA sequences distributed in the P. knowlesi genome. We designed and tested several novel primers specific to new target sequences in a single-tube, non-nested PCR assay and identified one set of primers that accurately detects P. knowlesi. We show that this primer set has 100% specificity for the detection of P. knowlesi using three different strains (Nuri, H, and Hackeri), and one human case of malaria caused by P. knowlesi. This test did not show cross reactivity with any of the four human malaria parasite species including 11 different strains of P. vivax as well as 5 additional species of simian malaria parasites.The new PCR assay based on novel P. knowlesi genomic sequence targets was able to accurately detect P. knowlesi. Additional laboratory and field-based testing of this assay will be necessary to further validate its utility for clinical diagnosis of P. knowlesi

Malaria parasite CelTOS targets the inner leaflet of cell membranes for pore- dependent disruption

Apicomplexan parasites contain a conserved protein CelTOS that, in malaria parasites, is essential for traversal of cells within the mammalian host and arthropod vector. However, the molecular role of CelTOS is unknown because it lacks sequence similarity to proteins of known function. Here, we determined the crystal structure of CelTOS and discovered CelTOS resembles proteins that bind to and disrupt membranes. In contrast to known membrane disruptors, CelTOS has a distinct architecture, specifically binds phosphatidic acid commonly present within the inner leaflet of plasma membranes, and potently disrupts liposomes composed of phosphatidic acid by forming pores. Microinjection of CelTOS into cells resulted in observable membrane damage. Therefore, CelTOS is unique as it achieves nearly universal inner leaflet cellular activity to enable the exit of parasites from cells during traversal. By providing novel molecular insight into cell traversal by apicomplexan parasites, our work facilitates the design of therapeutics against global pathogens. DOI: http://dx.doi.org/10.7554/eLife.20621.00