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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]

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]

Hepatitis B virus (HBV) infection is a liver disorder that can result in cirrhosis, liver failure and hepatocellular carcinoma. HBV infection remains a major global health problem, as it affects more 350 million people chronically and kills roughly 600,000 people annually. Drugs currently used against HBV include IFN-α that decreases viremia, inflammation and the growth of liver fibrosis, and adefovir that decreases the viral load. Each of these drugs can have severe side-effects. In the present paper, we consider the treatment of chronic HBV by a combination of IFN-α and adefovir, and raise the following question: What should be the optimal ratio between IFN-α and adefovir in order to achieve the best ‘efficacy’ under constraints on the total amount of the drugs; here the efficacy is measured by the reduction of the levels of inflammation and of fibrosis? We develop a mathematical model of HBV pathogenesis by a system of partial differential equations (PDEs) and use the model to simulate a ‘synergy map’ which addresses the above question. [ABSTRACT FROM AUTHOR]

Oncolytic virus (OV) is a replication competent virus that selectively invades cancer cells; as these cells die under the viral burden, the released virus particles proceed to infect other cancer cells. Oncolytic viruses are designed to also be able to stimulate the anticancer immune response. Thus, one may represent an OV by two parameters: its replication potential and its immunogenicity. In this paper we consider a combination therapy with OV and a checkpoint inhibitor, anti-PD-1. We evaluate the efficacy of the combination therapy in terms of the tumor volume at some later time, for example, 6 months from initial treatment. Since T cells kill not only virus-free cancer cells but also virus-infected cancer cells, the following question arises: Does increasing the amount of the checkpoint inhibitor always improve the efficacy? We address this question, by a mathematical model consisting of a system of partial differential equations. We use the model to construct, by simulations, an efficacy map in terms of the doses of the checkpoint inhibitor and the OV injection. We show that there are regions in the map where an increase in the checkpoint inhibitor actually decreases the efficacy of the treatment. We also construct efficacy maps with checkpoint inhibitor vs. the replication potential of the virus that show the same antagonism, namely, an increase in the checkpoint inhibitor may actually decrease the efficacy. These results have implications for clinical trials. [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]

Abdominal aortic aneurysm (AAA) is a localized enlargement of the abdominal aorta, such that the diameter exceeds 3 cm. The natural history of AAA is progressive growth leading to rupture, an event that carries up to 90% risk of mortality. Hence there is a need to predict the growth of the diameter of the aorta based on the diameter of a patient’s aneurysm at initial screening and aided by non-invasive biomarkers. IL-6 is overexpressed in AAA and was suggested as a prognostic marker for the risk in AAA. The present paper develops a mathematical model which relates the growth of the abdominal aorta to the serum concentration of IL-6. Given the initial diameter of the aorta and the serum concentration of IL-6, the model predicts the growth of the diameter at subsequent times. Such a prediction can provide guidance to how closely the patient’s abdominal aorta should be monitored. The mathematical model is represented by a system of partial differential equations taking place in the aortic wall, where the media is assumed to have the constituency of an hyperelastic material. [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]

Crohn’s Disease (CD) results from inappropriate response toward commensal flora. Earlier studies described CD as a Th1 mediated disease. Current models view both phenotypes as a continuum of various permutations between Th1, Th2 and Th17 pathways compounded by a range of Treg disfunctions. In the present paper, we develop a mathematical model, by a system of differential equations, which describe the dynamic relations among these T cells and their cytokines. The model identities four groups of CD patients according to up/down regulation of Th1 and Th2. The model simulations show that immunosuppression by TNF-α blockage benefits the group with Th1High/Th2Low while, by contrast, the group with Th1Low/Th2High will benefit from immune activation. [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]

Non-human primates are the animals closest to humans for use in influenza A virus challenge studies, in terms of their phylogenetic relatedness, physiology and immune systems. Previous studies have shown that cynomolgus macaques (Macaca fascicularis) are permissive for infection with H1N1pdm influenza virus. These studies have typically used combined challenge routes, with the majority being intra-tracheal delivery, and high doses of virus (> 107 infectious units). This paper describes the outcome of novel challenge routes (inhaled aerosol, intra-nasal instillation) and low to moderate doses (103 to 106 plaque forming units) of H1N1pdm virus in cynomolgus macaques. Evidence of virus replication and sero-conversion were detected in all four challenge groups, although the disease was sub-clinical. Intra-nasal challenge led to an infection confined to the nasal cavity. A low dose (103 plaque forming units) did not lead to detectable infectious virus shedding, but a 1000-fold higher dose led to virus shedding in all intra-nasal challenged animals. In contrast, aerosol and intra-tracheal challenge routes led to infections throughout the respiratory tract, although shedding from the nasal cavity was less reproducible between animals compared to the high-dose intra-nasal challenge group. Intra-tracheal and aerosol challenges induced a transient lymphopaenia, similar to that observed in influenza-infected humans, and greater virus-specific cellular immune responses in the blood were observed in these groups in comparison to the intra-nasal challenge groups. Activation of lung macrophages and innate immune response genes was detected at days 5 to 7 post-challenge. The kinetics of infection, both virological and immunological, were broadly in line with human influenza A virus infections. These more authentic infection models will be valuable in the determination of anti-influenza efficacy of novel entities against less severe (and thus more common) influenza infections. [ABSTRACT FROM AUTHOR]

There are currently over 2.5 million breast cancer survivors in the United States and, according to the American Cancer Society, 10 to 20 percent of these women will develop recurrent breast cancer. Early detection of recurrence can avoid unnecessary radical treatment. However, self-examination or mammography screening may not discover a recurring cancer if the number of surviving cancer cells is small, while biopsy is too invasive and cannot be frequently repeated. It is therefore important to identify non-invasive biomarkers that can detect early recurrence. The present paper develops a mathematical model of cancer recurrence. The model, based on a system of partial differential equations, focuses on tissue biomarkers that include the plasminogen system. Among them, only uPAR is known to have significant correlation to its concentration in serum and could therefore be a good candidate for serum biomarker. The model includes uPAR and other associated cytokines and cells. It is assumed that the residual cancer cells that survived primary cancer therapy are concentrated in the same location within a region with a very small diameter. Model simulations establish a quantitative relation between the diameter of the growing cancer and the total uPAR mass in the cancer. This relation is used to identify uPAR as a potential serum biomarker for breast cancer recurrence. [ABSTRACT FROM AUTHOR]

Glioblastoma is the most malignant and lethal form of astrocytoma, with patients having a median survival time of approximately 15 months with current therapeutic modalities. It is therefore important to identify novel therapeutics. There is mounting evidence that microglia (specialized brain-resident macrophages) play a significant role in the development and progression of glioblastoma tumors. In this paper we show that microglia, in addition to stimulating glioblastoma cell invasion, also promote glioblastoma cell proliferation and resistance to ionizing radiation in vitro. We found that semapimod, a drug that selectively interferes with the function of macrophages and microglia, potently inhibits microglia-stimulated GL261 invasion, without affecting serum-stimulated glioblastoma cell invasion. Semapimod also inhibits microglia-stimulated resistance of glioblastoma cells to radiation, but has no significant effect on microglia-stimulated glioblastoma cell proliferation. We also found that intracranially administered semapimod strongly increases the survival of GL261 tumor-bearing animals in combination with radiation, but has no significant benefit in the absence of radiation. In conclusion, our observations indicate that semapimod sensitizes glioblastoma tumors to ionizing radiation by targeting microglia and/or infiltrating macrophages. [ABSTRACT FROM AUTHOR]