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Established cell lines are widely used in research, however an appealing question is the comparability of the cells between various laboratories, their characteristics and stability in time. Problematic is also the cell line misidentification, genetic and phenotypic shift or Mycoplasma contamination which are often forgotten in research papers. The monocyte/macrophage-like cell line RAW 264.7 has been one of the most commonly used myeloid cell line for more than 40 years. Despite its phenotypic and functional stability is often discussed in literature or at various scientific discussion panels, their stability during the consecutive passages has not been confirmed in any solid study. So far, only a few functional features of these cells have been studied, for example their ability to differentiate into osteoclasts. Therefore, in the present paper we have investigated the phenotype and functional stability of the RAW 264.7 cell line from passage no. 5 till passage no. 50. We found out that the phenotype (expression of particular macrophage-characteristic genes and surface markers) and functional characteristics (phagocytosis and NO production) of RAW 264.7 cell line remains stable through passages: from passage no. 10 up to passage no. 30. Overall, our results indicated that the RAW 264.7 cell line should not be used after the passage no. 30 otherwise it may influence the data reliability. [ABSTRACT FROM AUTHOR]

The Epstein-Barr virus (EBV) induces B-cell proliferation with high efficiency through expression of latent proteins and microRNAs. This process takes place in vivo soon after infection, presumably to expand the virus reservoir, but can also induce pathologies, e.g. an infectious mononucleosis (IM) syndrome after primary infection or a B-cell lymphoproliferation in immunosuppressed individuals. In this paper, we investigated the growth characteristics of EBV-infected B-cells isolated from transplant recipients or patients with IM. We found that these cells grew and withstood apoptosis at highly variable rates, suggesting that the expansion rate of the infected B-cells widely varies between individuals, thereby influencing the size of the B-cell reservoir and the ability to form tumors in infected individuals. All viruses investigated were type 1 and genetically close to western strains. EBV-infected B-cells expressed the transforming EBV latent genes and microRNAs (miRNAs) at variable levels. We found that the B-cell growth rates positively correlated with the BHRF1 miRNA levels. Comparative studies showed that infected B-cells derived from transplant recipients with iEBVL on average expressed higher levels of EBV miR-BHRF1 miRNAs and grew more rapidly than B-cells from IM patients, suggesting infection by more transforming viruses. Altogether, these findings suggest that EBV infection has a highly variable impact on the B-cell compartment that probably reflects the genetic diversity of both the virus and the host. It also demonstrates the unexpected finding that B-cells from different individuals can grow at different speed under the influence of the same virus infection. [ABSTRACT FROM AUTHOR]

Blocking cytokine interleukin 10 (IL-10) at the time of immunisation enhances vaccine induced T cell responses and improves control of tumour cell growth in vivo. However, the effect of an IL-10 blockade on the biological function of macrophages has not been explored. In the current paper, a macrophage precursor cell line, U937 cells, was selected to investigate the differential expression of proteins and relevant cell signalling pathway changes, when stimulated with lipopolysaccharide (LPS) in the presence of antibodies to IL-10 or IL-10 receptor. We used a quantitative proteomic strategy to investigate variations in protein profiles of U937 cells following the treatments with LPS, LPS plus human anti-IL10 antibody and anti-IL10R antibody in 24hrs, respectively. The LPS treatment significantly activated actin-related cell matrix formation and immune response pathways. The addition of anti-IL10 and anti-IL10R antibody further promoted the immune response and potentially effect macrophage survival through PI3K/AKT signalling; however, the latter appeared to also upregulated oncogene XRCC5 and Cajal body associated processes. [ABSTRACT FROM AUTHOR]

B cells develop high affinity receptors during the course of affinity maturation, a cyclic process of mutation and selection. At the end of affinity maturation, a number of cells sharing the same ancestor (i.e. in the same “clonal family”) are released from the germinal center; their amino acid frequency profile reflects the allowed and disallowed substitutions at each position. These clonal-family-specific frequency profiles, called “substitution profiles”, are useful for studying the course of affinity maturation as well as for antibody engineering purposes. However, most often only a single sequence is recovered from each clonal family in a sequencing experiment, making it impossible to construct a clonal-family-specific substitution profile. Given the public release of many high-quality large B cell receptor datasets, one may ask whether it is possible to use such data in a prediction model for clonal-family-specific substitution profiles. In this paper, we present the method “Substitution Profiles Using Related Families” (SPURF), a penalized tensor regression framework that integrates information from a rich assemblage of datasets to predict the clonal-family-specific substitution profile for any single input sequence. Using this framework, we show that substitution profiles from similar clonal families can be leveraged together with simulated substitution profiles and germline gene sequence information to improve prediction. We fit this model on a large public dataset and validate the robustness of our approach on two external datasets. Furthermore, we provide a command-line tool in an open-source software package () implementing these ideas and providing easy prediction using our pre-fit models. [ABSTRACT FROM AUTHOR]

Purified recombinant proteins for use in biomedical research are invaluable to investigate protein function. However, purity varies in protein batches made in mammalian expression systems, such as CHO-cells or HEK293-cells. This study points to caution while investigating effects of proteins related to the transforming growth factor (TGF)-β superfamily. TGF-β itself is a very potent cytokine and has effects on cells in the femtomolar range. Thus, even very small amounts of contaminating TGF-β in purified protein batches may influence the experimental results given that receptors for TGF-β are present. When we attempted to characterize possible receptors for the TGF-β superfamily ligand GDF15, striking similarities between GDF15-induced activities and known TGF-β activities were found. However, differences between batches of GDF15 were a concern and finally led us to the conclusion that the measured effects were caused by TGF-β and not by GDF15. Our results emphasize that purified recombinant proteins must be used with caution and warrant proper controls. Notably, some conclusions made about GDF15 in already published papers may not be supported by the results shown. Awareness about this issue in the scientific community may prevent spreading of false positive results. [ABSTRACT FROM AUTHOR]

In the current era of big data, the amount of data available is continuously increasing. Both the number and types of samples, or features, are on the rise. The mixing of distinct features often makes interpretation more difficult. However, separate analysis of individual types requires subsequent integration. A tensor is a useful framework to deal with distinct types of features in an integrated manner without mixing them. On the other hand, tensor data is not easy to obtain since it requires the measurements of huge numbers of combinations of distinct features; if there are m kinds of features, each of which has N dimensions, the number of measurements needed are as many as Nm, which is often too large to measure. In this paper, I propose a new method where a tensor is generated from individual features without combinatorial measurements, and the generated tensor was decomposed back to matrices, by which unsupervised feature extraction was performed. In order to demonstrate the usefulness of the proposed strategy, it was applied to synthetic data, as well as three omics datasets. It outperformed other matrix-based methodologies. [ABSTRACT FROM AUTHOR]

Nucleoside diphosphate kinase (NDK), which has the same sequence as oncoprotein (OP) in humans, can induce nucleoside triphosphates in DNA replication by maintenance of the deoxynucleotide triphosphate (dNTP’s) and is known to be regulated by viral infection in the shrimp Litopenaeus vannamei. This paper describes the relationship between NDK and white spot syndrome virus (WSSV) infection. The recombinant NDK was produced by a prokaryotic expression system. WSSV copy numbers and mRNA levels of IE1 and VP28 were significantly increased in shrimp injected with recombinant NDK at 72 h after WSSV infection. After synthesizing dsRNA-NDK and confirming the efficacy of NDK silencing, we recorded the cumulative mortality of WSSV-infected shrimp injected with NDK and dsRNA-NDK. A comparison between the results demonstrated that silencing NDK delayed the death of shrimps. These findings indicate that NDK has an important role influencing the replication of WSSV replication in shrimp. Furthermore, NDK may have potential target as a new therapeutic strategy against WSSV infection in shrimp. [ABSTRACT FROM AUTHOR]

Background: Idiopathic pulmonary fibrosis (IPF) is a rare lung disease of unknown origin leading rapidly to death. This paper addresses the issue of whether sputum induction is a suitable tool to study respiratory tract inflammation and potential biomarkers in IPF compared to COPD, a fibrosing airway wall disease. Methods: In a cross-sectional analysis, 15 IPF patients, 32 COPD and 30 healthy subjects underwent sputum induction. Total sputum cell counts and the amount of TGF- β, IGF-1, IGF-2, IGFBP-1, IGFBP-2, IGFBP-3, IL-8, IL-13, MMP-7, MMP-9, YKL-40, TNF-α and KL-6 in sputum supernatant were analysed. We also profiled gene expression of cells in the induced sputum for TGF-β, MMP-7, YKL-40, IGFBP-2, IL-6, IL-8 and TNF-α. Results: IPF patients, like COPD, had increased sputum absolute number of neutrophils, eosinophils, macrophages and epithelial cells compared to HS. IPF sputum supernatants had increased concentrations of IGFBP-2, IL-8, TGF-β, MMP-7, MMP-9 and KL-6 (p<0.05, p<0.0001, p<0.05, p<0.05, p<0.0001, p<0.05 respectively) when compared to healthy subjects where COPD had higher IL-6 and TNF-α levels than IPF (p<0.05 and p<0.05 respectively) and HS (p<0.0001 and p<0.001 respectively) and higher IL-8 and MMP-9 than HS (p<0.0001 and p<0.001 respectively). Conversely to IL-6 and TNF-α, MMP-7 was increased in IPF compared to COPD (p<0.05). The KL-6 and MMP-7 protein levels in sputum were inversely correlated with total lung capacity (TLC, % of predicted) in IPF patients (r = -0.73 and r = -0.53 respectively). Sputum gene expression analysis identified a significant increase for IGFBP-2, IL-6, IL-8 and MMP-7 in IPF compared to HS (p<0.05, p<0.01, p<0.05 and p<0.0001 respectively) and for IGFBP-2, YKL-40, IL-6, IL-8 and MMP-7 compared to COPD (p<0.01, p<0.01, p<0.05, p<0.01 and p<0.0001 respectively). Furthermore, gene expression of TGF-β was increased in IPF compared to COPD (p<0.001) but not to HS. Conclusion: Our data show clear increase in expression and production of IGFBP-2, IL-8 and MMP-7 in sputum from patients with IPF that may contribute to the disease. [ABSTRACT FROM AUTHOR]

Paratuberculosis in cattle is a chronic granulomatous gastroenteritis caused by Mycobacterium avium subsp. paratubercolosis (MAP) which is endemic worldwide. In dairy herds, it is responsible for huge economic losses. However, current diagnostic methods do not detect subclinical infection making control of the disease difficult. The identification of MAP infected animals during the sub-clinical phase of infection would play a key role in preventing the dissemination of the pathogen and in reducing transmission. Gene expression and circulating microRNA (miRNA) signatures have been proposed as biomarkers of disease both in the human and veterinary medicine. In this paper, gene expression and related miRNA levels were investigated in cows positive for MAP, by ELISA and culture, in order to identify potential biomarkers to improve diagnosis of MAP infection. Three groups, each of 5 animals, were used to compare the results of gene expression from positive, exposed and negative cows. Overall 258 differentially expressed genes were identified between unexposed, exposed, but ELISA negative and positive groups which were involved in biological functions related to inflammatory response, lipid metabolism and small molecule biochemistry. Differentially expressed miRNA was also found among the three groups: 7 miRNAs were at a lower level and 2 at a higher level in positive animals vs unexposed animals, while 5 and 3 miRNAs were respectively reduced and increased in the exposed group compared to the unexposed group. Among the differentially expressed miRNAs 6 have been previously described as immune-response related and two were novel miRNAs. Analysis of the miRNA levels showed correlation with expression of their target genes, known to be involved in the immune process. This study suggests that miRNA expression is affected by MAP infection and play a key role in tuning the host response to infection. The miRNA and gene expression profiles may be biomarkers of infection and potential diagnostic of MAP infection earlier than the current ELISA based diagnostic tests. [ABSTRACT FROM AUTHOR]

The human immune system depends on a highly diverse collection of antibody-making B cells. B cell receptor sequence diversity is generated by a random recombination process called “rearrangement” forming progenitor B cells, then a Darwinian process of lineage diversification and selection called “affinity maturation.” The resulting receptors can be sequenced in high throughput for research and diagnostics. Such a collection of sequences contains a mixture of various lineages, each of which may be quite numerous, or may consist of only a single member. As a step to understanding the process and result of this diversification, one may wish to reconstruct lineage membership, i.e. to cluster sampled sequences according to which came from the same rearrangement events. We call this clustering problem “clonal family inference.” In this paper we describe and validate a likelihood-based framework for clonal family inference based on a multi-hidden Markov Model (multi-HMM) framework for B cell receptor sequences. We describe an agglomerative algorithm to find a maximum likelihood clustering, two approximate algorithms with various trade-offs of speed versus accuracy, and a third, fast algorithm for finding specific lineages. We show that under simulation these algorithms greatly improve upon existing clonal family inference methods, and that they also give significantly different clusters than previous methods when applied to two real data sets. [ABSTRACT FROM AUTHOR]

This paper distinguishes a rare subset of myeloid dendritic-like cells found in mouse spleen from conventional (c) dendritic cells (DC) in terms of phenotype, function and gene expression. These cells are tentatively named “L-DC” since they resemble dendritic-like cells produced in longterm cultures of spleen. L-DC can be distinguished on the basis of their unique phenotype as CD11bhiCD11cloMHCII-CD43+Ly6C-Ly6G-Siglec-F- cells. They demonstrate similar ability as cDC to uptake and retain complex antigens like mannan via mannose receptors, but much lower ability to endocytose and retain soluble antigen. While L-DC differ from cDC by their inability to activate CD4+ T cells, they are capable of antigen cross-presentation for activation of CD8+ T cells, although less effectively so than the cDC subsets. In terms of gene expression, CD8- cDC and CD8+ cDC are quite distinct from L-DC. CD8+ cDC are distinguishable from the other two subsets by expression of CD24a, Clec9a, Xcr1 and Tlr11, while CD8- cDC are distinguished by expression of Ccnd1 and H-2Eb2. L-DC are distinct from the two cDC subsets through upregulated expression of Clec4a3, Emr4, Itgam, Csf1r and CD300ld. The L-DC gene profile is quite distinct from that of cDC, confirming a myeloid cell type with distinct antigen presenting properties. [ABSTRACT FROM AUTHOR]

For the purposes of information retrieval, users must find highly relevant documents from within a system (and often a quite large one comprised of many individual documents) based on input query. Ranking the documents according to their relevance within the system to meet user needs is a challenging endeavor, and a hot research topic–there already exist several rank-learning methods based on machine learning techniques which can generate ranking functions automatically. This paper proposes a parallel B cell algorithm, RankBCA, for rank learning which utilizes a clonal selection mechanism based on biological immunity. The novel algorithm is compared with traditional rank-learning algorithms through experimentation and shown to outperform the others in respect to accuracy, learning time, and convergence rate; taken together, the experimental results show that the proposed algorithm indeed effectively and rapidly identifies optimal ranking functions. [ABSTRACT FROM AUTHOR]

The development and progression of systemic lupus erythematosus is mediated by the complex interaction of genetic and environmental factors. To decipher the genetics that contribute to pathogenesis and the production of pathogenic autoantibodies, our lab has focused on the generation of congenic lupus-prone mice derived from the New Zealand Black (NZB) strain. Previous work has shown that an NZB-derived chromosome 4 interval spanning 32 to 151 Mb led to expansion of CD5+ B and Natural Killer T (NKT) cells, and could suppress autoimmunity when crossed with a lupus-prone mouse strain. Subsequently, it was shown that CD5+ B cells but not NKT cells derived from these mice could suppress the development of pro-inflammatory T cells. In this paper, we aimed to further resolve the genetics that leads to expansion of these two innate-like populations through the creation of additional sub-congenic mice and to characterize the role of IL-10 in the suppression of autoimmunity through the generation of IL-10 knockout mice. We show that expansion of CD5+ B cells and NKT cells localizes to a chromosome 4 interval spanning 91 to 123 Mb, which is distinct from the region that mediates the majority of the suppressive phenotype. We also demonstrate that IL-10 is critical to restraining autoantibody production and surprisingly plays a vital role in supporting the expansion of innate-like populations. [ABSTRACT FROM AUTHOR]

During early human immunodeficiency virus (HIV) infection multiple CD8+ T cell responses are elicited almost simultaneously. These responses exert strong selective pressures on different parts of HIV’s genome, and select for mutations that escape recognition and are thus beneficial to the virus. Some studies reveal that the later these escape mutations emerge, the more slowly they go to fixation. This pattern of escape rate decrease(ERD) can arise by distinct mechanisms. In particular, in large populations with high beneficial mutation rates interference among different escape strains –an effect that can emerge in evolution with asexual reproduction and results in delayed fixation times of beneficial mutations compared to sexual reproduction– could significantly impact the escape rates of mutations. In this paper, we investigated how interference between these concurrent escape mutations affects their escape rates in systems with multiple epitopes, and whether it could be a source of the ERD pattern. To address these issues, we developed a multilocus Wright-Fisher model of HIV dynamics with selection, mutation and recombination, serving as a null-model for interference. We also derived an interference-free null model assuming initial neutral evolution before immune response elicitation. We found that interference between several equally selectively advantageous mutations can generate the observed ERD pattern. We also found that the number of loci, as well as recombination rates substantially affect ERD. These effects can be explained by the underexponential decline of escape rates over time. Lastly, we found that the observed ERD pattern in HIV infected individuals is consistent with both independent, interference-free mutations as well as interference effects. Our results confirm that interference effects should be considered when analyzing HIV escape mutations. The challenge in estimating escape rates and mutation-associated selective coefficients posed by interference effects cannot simply be overcome by improved sampling frequencies or sizes. This problem is a consequence of the fundamental shortcomings of current estimation techniques under interference regimes. Hence, accounting for the stochastic nature of competition between mutations demands novel estimation methodologies based on the analysis of HIV strains, rather than mutation frequencies. [ABSTRACT FROM AUTHOR]

Variable region-containing chitin-binding proteins (VCBPs) are secreted, immune-type molecules that have been described in both amphioxus, a cephalochordate, and sea squirt, Ciona intestinalis, a urochordate. In adult Ciona, VCBP-A, -B and -C are expressed in hemocytes and the cells of the gastrointestinal tract. VCBP-C binds bacteria in the stomach lumen and functions as an opsonin in vitro. In the present paper the expression of VCBPs has been characterized during development using in situ hybridization, immunohistochemical staining and quantitative polymerase chain reaction (qPCR) technologies. The expression of VCBP-A and -C is detected first in discrete areas of larva endoderm and becomes progressively localized during differentiation in the stomach and intestine, marking the development of gut tracts. In “small adults” (1–2 cm juveniles) expression of VCBP-C persists and VCBP-A gradually diminishes, ultimately replaced by expression of VCBP-B. The expression of VCBP-A and -C in stage 7–8 juveniles, at which point animals have already started feeding, is influenced significantly by challenge with either Gram-positive or -negative bacteria. A potential role for VCBPs in gut-microbiota interactions and homeostasis is indicated. [ABSTRACT FROM AUTHOR]

Primary dysmenorrhea is one of the most common gynecological complaints in young women, but potential peripheral immunologic features underlying this condition remain undefined. In this paper, we compared 84 common cytokine gene expression profiles of peripheral blood mononuclear cells (PBMCs) from six primary dysmenorrheic young women and three unaffected controls on the seventh day before (secretory phase), and the first (menstrual phase) and the fifth (regenerative phase) days of menstruation, using a real-time PCR array assay combined with pattern recognition and gene function annotation methods. Comparisons between dysmenorrhea and normal control groups identified 11 (nine increased and two decreased), 14 (five increased and nine decreased), and 15 (seven increased and eight decreased) genes with ≥2-fold difference in expression (P<0.05) in the three phases of menstruation, respectively. In the menstrual phase, genes encoding pro-inflammatory cytokines (IL1B, TNF, IL6, and IL8) were up-regulated, and genes encoding TGF-β superfamily members (BMP4, BMP6, GDF5, GDF11, LEFTY2, NODAL, and MSTN) were down-regulated. Functional annotation revealed an excessive inflammatory response and insufficient TGF-β superfamily member signals with anti-inflammatory consequences, which may directly contribute to menstrual pain. In the secretory and regenerative phases, increased expression of pro-inflammatory cytokines and decreased expression of growth factors were also observed. These factors may be involved in the regulation of decidualization, endometrium breakdown and repair, and indirectly exacerbate primary dysmenorrhea. This first study of cytokine gene expression profiles in PBMCs from young primary dysmenorrheic women demonstrates a shift in the balance between expression patterns of pro-inflammatory cytokines and TGF-β superfamily members across the whole menstrual cycle, underlying the peripheral immunologic features of primary dysmenorrhea. [ABSTRACT FROM AUTHOR]