Hemocyte-lineage marker proteins in a crustacean, the freshwater crayfish, Pacifastacus leniusculus

To identify proteins associated with development of different hemocyte types in the freshwater crayfish Pacifastacus leniusculus, 2-DE followed by MS analysis was carried out with hematopoietic tissue (Hpt) cells, semigranular cells (SGC) and granular cells (GC). Within the hemocyte lineages one two-domain Kazal proteinase inhibitor (KPI) was found to be specific,for SGC, while a superoxide dismutase (SOD) was specific for GC at protein as well as at mRNA level. The proliferation cell nuclear antigen (PCNA) was detected at the mRNA level in Hpt cells only. We also provide evidence that SGC and GC most likely differentiate to maturation as separate lineages. We found that after laminarin or lipopolysaccharide (LPS) injection into crayfish, the transcript levels of PCNA and SOD increased in the Hpt cells, whereas the KPI transcript never was present in Hpt regardless of any challenge. RNA interference of PCNA in the Hpt cells led to that most of the cells did not spread or attach to the tissue culture dish. These results suggest that PCNA, KPI and SOD can be used as markers for Hpt cells, SGC and GC, respectively, and in conjunction with these results, a model is proposed how the Hpt responds to a microbial challenge by proliferation and release of Hpt cells

Histone H2A as a transfection agent in crayfish hematopoietic tissue cells

We report a novel and highly efficient dsRNA transfection system based on one of the nuclear proteins, namely, histone H2A. RT-PCR semi-quantitative analysis of silencing target gene shows that the transfection efficiency of histone H2A is higher than Effectene or liposome-based transfection systems. Importantly, the high efficiency of histone H2A was associated with very low toxicity to the transfected crayfish hematopoietic tissue (Hpt) cells. The non-toxicity, effectiveness and specificity of histone H2A as a transfection agent provides a cheap, simple, highly efficient and reproducible gene delivery system, particularly for the sensitive cell cultures of crustacean animals such as crayfish and shrimp. (c) 2006 Elsevier Ltd. All rights reserved

A pooling-based genome-wide analysis identifies new potential candidate genes for atopy in the European Community Respiratory Health Survey (ECRHS)

<p>Abstract</p> <p>Background</p> <p>Asthma and atopy are complex phenotypes with shared genetic component. In this study we attempt to identify genes related to these traits performing a two-stage DNA pooling genome-wide analysis in order to reduce costs. First, we assessed all markers in a subset of subjects using DNA pooling, and in a second stage we evaluated the most promising markers at an individual level.</p> <p>Methods</p> <p>For the genome-wide analysis, we constructed DNA pools from 75 subjects with atopy and asthma, 75 subjects with atopy and without asthma and 75 control subjects without atopy or asthma. In a second stage, the most promising regions surrounding significant markers after correction for false discovery rate were replicated with individual genotyping of samples included in the pools and an additional set of 429 atopic subjects and 222 controls from the same study centres.</p> <p>Results</p> <p><it>Homo sapiens </it>protein kinase-like protein SgK493 (<it>SGK493</it>) was found to be associated with atopy. To lesser extent mitogen-activated protein kinase 5 (<it>MAP3K5</it>), collagen type XVIII alpha 1 (<it>COL18A1</it>) and collagen type XXIX alpha 1 (<it>COL29A1</it>) were also found to be associated with atopy. Functional evidences points out a role for <it>MAP3K5</it>, <it>COL18A1 </it>and <it>COL29A1 </it>but the function of <it>SGK493 </it>is unknown.</p> <p>Conclusion</p> <p>In this analysis we have identified new candidate regions related to atopy and suggest <it>SGK493 </it>as an atopy locus, although these results need further replication.</p

Immunomodulation by Different Types of N-Oxides in the Hemocytes of the Marine Bivalve Mytilus galloprovincialis

The potential toxicity of engineered nanoparticles (NPs) for humans and the environment represents an emerging issue. Since the aquatic environment represents the ultimate sink for NP deposition, the development of suitable assays is needed to evaluate the potential impact of NPs on aquatic biota. The immune system is a sensitive target for NPs, and conservation of innate immunity represents an useful basis for studying common biological responses to NPs. Suspension-feeding invertebrates, such as bivalves, are particularly at risk to NP exposure, since they have extremely developed systems for uptake of nano and microscale particles integral to intracellular digestion and cellular immunity. Evaluation of the effects of NPs on functional parameters of bivalve immunocytes, the hemocytes, may help understanding the major toxic mechanisms and modes of actions that could be relevant for different NP types in aquatic organisms.In this work, a battery of assays was applied to the hemocytes of the marine bivalve Mytilus galloprovincialis to compare the in vitro effects of different n-oxides (n-TiO2, n-SiO2, n-ZnO, n-CeO2) chosen on the basis of their commercial and environmental relevance. Physico-chemical characterization of both primary particles and NP suspensions in artificial sea water-ASW was performed. Hemocyte lysosomal and mitochondrial parameters, oxyradical and nitric oxide production, phagocytic activity, as well as NP uptake, were evaluated. The results show that different n-oxides rapidly elicited differential responses hemocytes in relation to their chemical properties, concentration, behavior in sea water, and interactions with subcellular compartments. These represent the most extensive data so far available on the effects of NPs in the cells of aquatic organisms. The results indicate that Mytilus hemocytes can be utilized as a suitable model for screening the potential effects of NPs in the cells of aquatic invertebrates, and may provide a basis for future experimental work for designing environmentally safer nanomaterials

Prenatal particulate air pollution and DNA methylation in newborns: An epigenome-wide meta-analysis

BACKGROUND: Prenatal exposure to air pollution has been associated with childhood respiratory disease and other adverse outcomes. Epigenetics is a suggested link between exposures and health outcomes. OBJECTIVES: We aimed to investigate associations between prenatal exposure to particulate matter (PM) with diameter &lt;10 (PM 10)or&lt;2:5 lm (PM 2:5) and DNA methylation in newborns and children. METHODS: We meta-analyzed associations between exposure to PM 10 (n = 1,949) and PM 2:5 (n = 1,551) at maternal home addresses during pregnancy and newborn DNA methylation assessed by Illumina Infinium HumanMethylation450K BeadChip in nine European and American studies, with replication in 688 independent newborns and look-up analyses in 2,118 older children. We used two approaches, one focusing on single cytosine-phosphate-guanine (CpG) sites and another on differentially methylated regions (DMRs). We also related PM exposures to blood mRNA expression. RESULTS: Six CpGs were significantly associated [false discovery rate (FDR) &lt;0:05] with prenatal PM 10 and 14 with PM 2:5 exposure. Two of the PM 10-related CpGs mapped to FAM13A (cg00905156) and NOTCH4 (cg06849931) previously associated with lung function and asthma. Although these associations did not replicate in the smaller newborn sample, both CpGs were significant (p &lt;0:05) in 7-to 9-y-olds. For cg06849931, however, the direction of the association was inconsistent. Concurrent PM 10 exposure was associated with a significantly higher NOTCH4 expression at age 16 y. We also identified several DMRs associated with either prenatal PM 10 and or PM 2:5 exposure, of which two PM 10-related DMRs, including H19 and MARCH11, replicated in newborns. CONCLUSIONS: Several differentially methylated CpGs and DMRs associated with prenatal PM exposure were identified in newborns, with annotation to genes previously implicated in lung-related outcomes. https://doi.org/10.1289/EHP4522. </p

Involvement of the Cytokine MIF in the Snail Host Immune Response to the Parasite Schistosoma mansoni

We have identified and characterized a Macrophage Migration Inhibitory Factor (MIF) family member in the Lophotrochozoan invertebrate, Biomphalaria glabrata, the snail intermediate host of the human blood fluke Schistosoma mansoni. In mammals, MIF is a widely expressed pleiotropic cytokine with potent pro-inflammatory properties that controls cell functions such as gene expression, proliferation or apoptosis. Here we show that the MIF protein from B. glabrata (BgMIF) is expressed in circulating immune defense cells (hemocytes) of the snail as well as in the B. glabrata embryonic (Bge) cell line that has hemocyte-like features. Recombinant BgMIF (rBgMIF) induced cell proliferation and inhibited NO-dependent p53-mediated apoptosis in Bge cells. Moreover, knock-down of BgMIF expression in Bge cells interfered with the in vitro encapsulation of S. mansoni sporocysts. Furthermore, the in vivo knock-down of BgMIF prevented the changes in circulating hemocyte populations that occur in response to an infection by S. mansoni miracidia and led to a significant increase in the parasite burden of the snails. These results provide the first functional evidence that a MIF ortholog is involved in an invertebrate immune response towards a parasitic infection and highlight the importance of cytokines in invertebrate-parasite interactions

Gene–Environment Interaction Affects Risk of Atopic Eczema: Population and In Vitro Studies

\ua9 2025 The Author(s). Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley &amp; Sons Ltd.Background: Multiple environmental and genetic factors play a role in the pathogenesis of atopic eczema (AE). We aimed to investigate gene–environment interactions (G 7 E) to improve understanding of the pathophysiology. Methods: We analysed data from 16 European studies to test for interaction between the 24 most significant AE-associated loci identified from genome-wide association studies and 18 early-life environmental factors. We tested for replication using a further 10 studies and in vitro modeling to independently assess findings. Results: The discovery analysis (including 25,339 individuals) showed suggestive evidence for interaction (p &lt; 0.05) between seven environmental factors (antibiotic use, cat ownership, dog ownership, breastfeeding, elder sibling, smoking and washing practices) and at least one established variant for AE, 14 interactions in total. In the replication analysis (254,532 individuals) dog exposure 7 rs10214237 (on chromosome 5p13.2 near IL7R) was nominally significant (ORinteraction = 0.91 [0.83–0.99] p = 0.025), with a risk effect of the T allele observed only in those not exposed to dogs. A similar interaction with rs10214237 was observed for siblings in the discovery analysis (ORinteraction = 0.84 [0.75–0.94] p = 0.003), but replication analysis was under-powered (ORinteraction = 1.09 [0.82–1.46]). rs10214237 homozygous risk genotype is associated with lower IL-7R expression in human keratinocytes, and dog exposure modelled in vitro showed a differential response according to rs10214237 genotype. Conclusion: Interaction analysis and functional assessment provide preliminary evidence that early-life dog exposure may modify the genetic effect of rs10214237 on AE via IL7R, supporting observational epidemiology showing a protective effect for dog ownership. The lack of evidence for other G 7 E studied here implies only weak effects are likely to occur

Conidiation Color Mutants of Aspergillus fumigatus Are Highly Pathogenic to the Heterologous Insect Host Galleria mellonella

The greater wax moth Galleria mellonella has been widely used as a heterologous host for a number of fungal pathogens including Candida albicans and Cryptococcus neoformans. A positive correlation in pathogenicity of these yeasts in this insect model and animal models has been observed. However, very few studies have evaluated the possibility of applying this heterologous insect model to investigate virulence traits of the filamentous fungal pathogen Aspergillus fumigatus, the leading cause of invasive aspergillosis. Here, we have examined the impact of mutations in genes involved in melanin biosynthesis on the pathogenicity of A. fumigatus in the G. mellonella model. Melanization in A. fumigatus confers bluish-grey color to conidia and is a known virulence factor in mammal models. Surprisingly, conidial color mutants in B5233 background that have deletions in the defined six-gene cluster required for DHN-melanin biosynthesis caused enhanced insect mortality compared to the parent strain. To further examine and confirm the relationship between melanization defects and enhanced virulence in the wax moth model, we performed random insertional mutagenesis in the Af293 genetic background to isolate mutants producing altered conidia colors. Strains producing conidia of previously identified colors and of novel colors were isolated. Interestingly, these color mutants displayed a higher level of pathogenicity in the insect model compared to the wild type. Although some of the more virulent color mutants showed increased resistance to hydrogen peroxide, overall phenotypic characterizations including secondary metabolite production, metalloproteinase activity, and germination rate did not reveal a general mechanism accountable for the enhanced virulence of these color mutants observed in the insect model. Our observations indicate instead, that exacerbated immune response of the wax moth induced by increased exposure of PAMPs (pathogen-associated molecular patterns) may cause self-damage that results in increased mortality of larvae infected with the color mutants. The current study underscores the limitations of using this insect model for inferring the pathogenic potential of A. fumigatus strains in mammals, but also points to the importance of understanding the innate immunity of the insect host in providing insights into the pathogenicity level of different fungal strains in this model. Additionally, our observations that melanization defective color mutants demonstrate increased virulence in the insect wax moth, suggest the potential of using melanization defective mutants of native insect fungal pathogens in the biological control of insect populations

Moult cycle specific differential gene expression profiling of the crab Portunus pelagicus

Background: Crustacean moulting is a complex process involving many regulatory pathways. A holistic approach to examine differential gene expression profiles of transcripts relevant to the moulting process, across all moult cycle stages, was used in this study. Custom cDNA microarrays were constructed for Portunus pelagicus. The printed arrays contained 5000 transcripts derived from both the whole organism, and from individual organs such as the brain, eyestalk, mandibular organ and Y-organ from all moult cycle stages.Results: A total of 556 clones were sequenced from the cDNA libraries used to construct the arrays. These cDNAs represented 175 singletons and 62 contigs, resulting in 237 unique putative genes. The gene sequences were classified into the following biological functions: cuticular proteins associated with arthropod exoskeletons, farnesoic acid O-methyltransferase (FaMeT), proteins belonging to the hemocyanin gene family, lectins, proteins relevant to lipid metabolism, mitochondrial proteins, muscle related proteins, phenoloxidase activators and ribosomal proteins. Moult cycle-related differential expression patterns were observed for many transcripts. Of particular interest were those relating to the formation and hardening of the exoskeleton, and genes associated with cell respiration and energy metabolism.Conclusions: The expression data presented here provide a chronological depiction of the molecular events associated with the biological changes that occur during the crustacean moult cycle. Tracing the temporal expression patterns of a large variety of transcripts involved in the moult cycle of P. pelagicus can provide a greater understanding of gene function, interaction, and regulation of both known and new genes with respect to the moulting process