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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]

Triticum vulgare has been extensively used in traditional medicine thanks to its properties of accelerating tissue repair. The specific extract of Triticum vulgare manufactured by Farmaceutici Damor (TVE-DAMOR) is already present in some pharmaceutical formulations used in the treatment of decubitus ulcers, skin lesions and burns. It has been recently suggested that this Triticum vulgare extract may possess potential anti-inflammatory properties. In the light of these premises the aim of the present paper was to verify the anti-inflammatory role of TVE, using the LPS-stimulated microglia model of inflammation. In particular the effect of different concentrations of TVE on the release of several mediators of inflammation such as nitric oxide, IL-6, PGE2 and TNF alpha was evaluated. More important, the anti-inflammatory effect of TVE was confirmed also in primary rat microglia cultures. The results of the present study show that TVE exerts anti-inflammatory properties since it reduces the release of all the evaluated markers of inflammation, such as NO, IL6, TNF alpha and PGE2 in LPS-activated BV2 microglial cells. Intriguingly, TVE reduced microglia activation and NO release also in primary microglia. Indeed, to verify the pathway of modulation of the inflammatory markers reported above, we found that TVE restores the cytoplasmic expression of p65 protein, kwown as specific marker associated with activation of inflammatory response. The evidence for an inhibitory activity on inflammation of this specific extract of Triticum vulgare may open the way to the possibility of a therapeutical use of the Triticum vulgare extract as an anti-inflammatory compound in certain pathological states such as burns, decubitus ulcers, folliculitis and inflammation of peripheral nerve. [ABSTRACT FROM AUTHOR]

Due to its strong proliferation-reducing effects on keratinocytes, and also anti-inflammatory properties, the isoflavone genistein has already been proposed as a possible antipsoriatic compound. As there is still no detailed information on this topic, we examined the effects of genistein by using an in vitro model of both, normal and “psoriasis-like” keratinocytes at this stage of our work exhaustively testing the selected flavonoid in a mono-treated experimental design. Gene expression studies revealed transcriptional changes that confirms known disease-associated pathways and highlights many psoriasis-related genes. Our results suggested that aberrant expression of genes contributing to the progress of psoriasis could be improved by the action of genistein. Genistein prevented “cytokine mix” as well as TNF-α-induced NF-κB nuclear translocation, with no effect on the PI3K signaling cascade, indicating the luck of turning this pathway into NF-κB activation. It could have attenuated TNF-α and LPS-induced inflammatory responses by suppressing ROS activation. Regardless of the type of keratinocyte stimulation used, reduction of cytokine IL-8, IL-20 and CCL2 production (both at RNA and protein level) following genistein treatment was visible. Because investigations of other groups supported our commentary on potential administration of genistein as a potential weapon in the armamentarium against psoriasis, it is believed that this paper should serve to encourage researchers to conduct further studies on this subject. [ABSTRACT FROM AUTHOR]

Rottlerin is a natural polyphenolic compound that was initially indicated as a PKCδ inhibitor. However, it was recently revealed that it may target a number of molecules and have biological effects on various cell types and is considered as a possible agent for tumor and cell proliferative diseases. Psoriasis is a chronic inflammatory cutaneous disorder with undefined etiology and is characterized by abnormal cellular proliferation, angiogenesis, and inflammation. Therefore, this paper investigates the regulatory effects of rottlerin on normal human epidermal keratinocytes (NHEKs) and imiquimod (IMQ)-induced psoriasiform (IPI) lesions. In vitro results showed that rottlerin inhibited cell proliferation in NHEKs through growth arrest and NFκB inhibition. It may also induce apoptosis in an autophagy-dependent pathway. We found that rottlerin inhibited human microvascular endothelial cells tube formation on matrigel. Rottlerin also decreased the cell senescence of keratinocytes and intracellular ROS generation, which indicated its antioxidant effect. We also showed that rottlerin affects the expression of keratinocyte proliferation biomarkers. In 12-O-tetradecanoylphorbol13-acetate (TPA)–induced keratinocytes, rottlerin significantly inhibited the expression of the induced pro-inflammatory cytokines in keratinocytes. An animal experiment provided the corresponding evidence based on this evidence in vitro, by using IPI model, we found that rottlerin could relieve the psoriasiform of BALB/c mice by inhibiting keratinocyte proliferation, inflammatory cell infiltration, and vascular proliferation. In conclusion, our results suggest that rottlerin may prove useful in the development of therapeutic agents against psoriasis. However, the deep mechanism still requires further study. [ABSTRACT FROM AUTHOR]

T-cells and antigen presenting cells are an essential part of the adaptive immune response system and how they interact is crucial in how the body effectively fights infection or responds to vaccines. Much of the experimental work studying interaction forces between cells has looked at the average properties of bulk samples of cells or applied microscopy to image the dynamic contact between these cells. In this paper we present a novel optical trapping technique for interrogating the force of this interaction and measuring relative interaction forces at the single-cell level. A triple-spot optical trap is used to directly manipulate the cells of interest without introducing foreign bodies such as beads to the system. The optical trap is used to directly control the initiation of cell-cell contact and, subsequently to terminate the interaction at a defined time point. The laser beam power required to separate immune cell pairs is determined and correlates with the force applied by the optical trap. As proof of concept, the antigen-specific increase in interaction force between a dendritic cell and a specific T-cell is demonstrated. Furthermore, it is demonstrated that this interaction force is completely abrogated when T-cell signalling is blocked. As a result the potential of using optical trapping to interrogate cellular interactions at the single cell level without the need to introduce foreign bodies such as beads is clearly demonstrated. [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]

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]

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]

The expression of cancer stemness is believed to reduce the efficacy of current therapies against hepatocellular carcinoma (HCC). Understanding of the stemness-regulating signaling pathways incurred by a specific etiology can facilitate the development of novel targets for individualized therapy against HCC. Niche environments, such as virus-induced inflammation, may play a crucial role. However, the mechanisms linking inflammation and stemness expression in HCC remain unclear. Here we demonstrated the distinct role of inflammatory mediators in expressions of stemness-related properties involving the pluripotent octamer-binding transcription factor 4 (OCT4) in cell migration and drug resistance of hepatitis B virus-related HCC (HBV-HCC). We observed positive immunorecognition for macrophage chemoattractant protein 1 (MCP-1)/CD68 and OCT4/NANOG in HBV-HCC tissues. The inflammation-conditioned medium (inflamed-CM) generated by lipopolysaccharide-stimulated U937 human leukemia cells significantly increased the mRNA and protein levels of OCT4/NANOG preferentially in HBV-active (HBV+HBsAg+) HCC cells. The inflamed-CM also increased the side population (SP) cell percentage, green fluorescent protein (GFP)-positive cell population, and luciferase activity of OCT4 promoter-GFP/luciferase in HBV-active HCC cells. Furthermore, the inflamed-CM upregulated the expressions of insulin-like growth factor-I (IGF-I)/IGF-I receptor (IGF-IR) and activated IGF-IR/Akt signaling in HBV-HCC. The IGF-IR phosphorylation inhibitor picropodophyllin (PPP) suppressed inflamed-CM-induced OCT4 and NANOG levels in HBV+HBsAg+ Hep3B cells. Forced expression of OCT4 significantly increased the secondary sphere formation and cell migration, and reduced susceptibility of HBV-HCC cells to cisplatin, bleomycin, and doxorubicin. Taking together, our results show that niche inflammatory mediators play critical roles in inducing the expression of stemness-related properties involving IGF-IR activation, and the upregulation of OCT4 contributes to cancer migration and drug resistance of HBV-HCC cells. Findings in this paper would provide potential targets for a therapeutic strategy targeting on inflammatory environment for HBV-HCC. [ABSTRACT FROM AUTHOR]

Enhancement or inhibition of cytokine signaling and corresponding immune cells responses are critical factors in various disease treatments. Cytokine signaling may be inhibited by cytokine-neutralizing antibodies (CNAs), which prevents further activation of cytokine receptors. However, CNAs may result in enhanced—instead of inhibitory—cytokine signaling (an “agonistic effect”) in various in vitro and in vivo experiments. This may lead to lack of efficacy or adverse events for cytokine-inhibiting based medicines. Alternatively, cytokine-antibody complexes may produce stronger signaling vs. cytokine alone, thereby increasing the efficacy of stimulating cytokine-based drugs, at equal or lower cytokine doses. In this paper, the effect of cytokine signaling enhancement by a CNA was studied in a generic mathematical model of interleukin-4 (IL-4) driven T-cell proliferation. The occurrence of the agonistic effect depends upon the antibody-to-cytokine binding affinity and initial concentrations of antibody and cytokine. Model predictions were in agreement with experimental studies. When the cytokine receptor consists of multiple subunits with substantially differing affinities (e.g., IL-4 case), the choice of the receptor chain to be blocked by the antibody is critical, for the agonistic effect to appear. We propose a generic mechanism for the effect: initially, binding of the CNA to the cytokine reduces free cytokine concentration; yet, cytokine molecules bound within the cytokine-CNA complex—and released later and over time—are “rescued” from earlier clearance via cellular internalization. Hence, although free cytokine-dependent signalling may be less potent initially, it will also be more sustained over time; and given non-linear dynamics, it will lead ultimately to larger cellular effector responses, vs. the same amount of free cytokine in the absence of CNA. We suggest that the proposed mechanism is a generic property of {cytokine, CNA, receptor} triads, both in vitro and in vivo, and can occur in a predictable fashion for a variety of cytokines of the immune system. [ABSTRACT FROM AUTHOR]

The spread of KPC-type carbapenemases is mainly attributed to the global dissemination of Klebsiella pneumoniae (KP) strains belonging to the clonal group (CG) 258, including sequence type (ST) 258 and other related STs. Two distinct clades of CG258-KP have evolved, which differ mainly for the composition of their capsular polysaccharides, and recent studies indicate that clade 1 evolved from an ancestor of clade 2 by recombination of a genomic fragment carrying the capsular polysaccharide (cps) locus. In this paper, we investigated the ability of two ST258-KP strains, KKBO-1 and KK207-1, selected as representatives of ST258-KP clade 2 and clade 1, respectively, to activate an adaptive immune response using ex vivo-stimulation of PBMC from normal donors as an experimental model. Our data showed that KKBO-1 (clade 2) induces a Th17 response more efficiently than KK207-1 (clade 1): the percentage of CD4+IL17+ cells and the production of IL-17A were significantly higher in cultures with KKBO-1 compared to cultures with KK207-1. While no differences in the rate of bacterial internalization or in the bacteria-induced expression of CD86 and HLA-DR by monocytes and myeloid dendritic cells were revealed, we found that the two strains significantly differ in inducing the production of cytokines involved in the adaptive immune response, as IL-1β, IL-23 and TNF-α, by antigen-presenting cells, with KKBO-1 being a more efficient inducer than KK207-1. The immune responses elicited by KK207-1 were comparable to those elicited by CIP 52.145, a highly virulent K. pneumoniae reference strain known to escape immune-inflammatory responses. Altogether, present results suggest that CG258-KP of the two clades are capable of inducing a different response of adaptive immunity in the human host. [ABSTRACT FROM AUTHOR]

Anaplasma marginale is the most prevalent tick-borne livestock pathogen and poses a significant threat to cattle industry. In contrast to currently available live blood-derived vaccines against A. marginale, alternative safer and better-defined subunit vaccines will be of great significance. Two proteins (VirB9-1 and VirB9-2) from the Type IV secretion system of A. marginale have been shown to induce humoral and cellular immunity. In this study, Escherichia coli were used to express VirB9-1 and VirB9-2 proteins. Silica vesicles having a thin wall of 6 nm and pore size of 5.8 nm were used as the carrier and adjuvant to deliver these two antigens both as individual or mixed nano-formulations. High loading capacity was achieved for both proteins, and the mouse immunisation trial with individual as well as mixed nano-formulations showed high levels of antibody titres over 107 and strong T-cell responses. The mixed nano-formulation also stimulated high-level recall responses in bovine T-cell proliferation assays. These results open a promising path towards the development of efficient A. marginale vaccines and provide better understanding on the role of silica vesicles to deliver multivalent vaccines as mixed nano-formulations able to activate both B-cell and T-cell immunity, for improved animal health. [ABSTRACT FROM AUTHOR]

It is common to find abundant genetic variation in host resistance and parasite infectivity within populations, with the outcome of infection frequently depending on genotype-specific interactions. Underlying these effects are complex immune defenses that are under the control of both host and parasite genes. We have found extensive variation in Drosophila melanogaster’s immune response against the parasitoid wasp Leptopilina boulardi. Some aspects of the immune response, such as phenoloxidase activity, are predominantly affected by the host genotype. Some, such as upregulation of the complement-like protein Tep1, are controlled by the parasite genotype. Others, like the differentiation of immune cells called lamellocytes, depend on the specific combination of host and parasite genotypes. These observations illustrate how the outcome of infection depends on independent genetic effects on different aspects of host immunity. As parasite-killing results from the concerted action of different components of the immune response, these observations provide a physiological mechanism to generate phenomena like epistasis and genotype-interactions that underlie models of coevolution. [ABSTRACT FROM AUTHOR]

Eosinophils are the prominent inflammatory cell involved in allergic asthma, atopic dermatitis, eosinophilic esophagitis, and hypereosinophilic syndrome and are found in high numbers in local tissue and/or circulating blood of affected patients. There is recent interest in a family of alarmins, including TSLP, IL-25 and IL-33, that are epithelial-derived and released upon stimulation of epithelial cells. Several genome wide association studies have found SNPs in genes encoding IL-33 to be risk factors for asthma. In two studies examining the direct role of IL-33 in eosinophils, there were differences in eosinophil responses. We sought to further characterize activation of eosinophils with IL-33 compared to activation by other cytokines and chemokines. We assessed IL-33 stimulated adhesion, degranulation, chemotaxis and cell surface protein expression in comparison to IL-3, IL-5, and eotaxin-1 on human eosinophils. Our results demonstrate that IL-33 can produce as potent eosinophil activation as IL-3, IL-5 and eotaxin-1. Thus, when considering specific cytokine targeting strategies, IL-33 will be important to consider for modulating eosinophil function. [ABSTRACT FROM AUTHOR]

Escherichia coli infection of the female reproductive tract is a significant cause of disease in humans and animals, but simple animal models are lacking. Here we report that vaginal inoculation of uropathogenic E. coli strains UTI89 and CFT073 in non-pregnant, estrogen-treated mice resulted in robust colonization of the vagina and uterine horns, whereas titers of the lab strain MG1655 were significantly lower. Non-estrogenized mice also became colonized, but there was more variation in titers. A dose of 104 colony-forming units (CFU) UTI89 was sufficient to result in colonization in all estrogenized mice, and we also observed bacterial transfer between inoculated and uninoculated estrogenized cage mates. UTI89 infection led to inflammation and leukocyte infiltration into the uterine horns as evidenced by tissue histology. Flow cytometry experiments revealed that neutrophil, monocyte and eosinophil populations were significantly increased in infected uterine horns. This model is a simple way to study host-pathogen interactions in E. coli vaginal colonization and uterine infection. There are immediate implications for investigators studying urinary tract infection using mouse models, as few E. coli are required to achieve reproductive colonization, resulting in an additional, underappreciated mucosal reservoir. [ABSTRACT FROM AUTHOR]

Hypervariable T cell receptors (TCRs) play a key role in adaptive immunity, recognizing a vast diversity of pathogen-derived antigens. Our ability to extract clinically relevant information from large high-throughput sequencing of TCR repertoires (RepSeq) data is limited, because little is known about TCR–disease associations. We present Antigen-specific Lymphocyte Identification by Clustering of Expanded sequences (ALICE), a statistical approach that identifies TCR sequences actively involved in current immune responses from a single RepSeq sample and apply it to repertoires of patients with a variety of disorders—patients with autoimmune disease (ankylosing spondylitis [AS]), under cancer immunotherapy, or subject to an acute infection (live yellow fever [YF] vaccine). We validate the method with independent assays. ALICE requires no longitudinal data collection nor large cohorts, and it is directly applicable to most RepSeq datasets. Its results facilitate the identification of TCR variants associated with diseases and conditions, which can be used for diagnostics and rational vaccine design. [ABSTRACT FROM AUTHOR]

Background: Hypercholesterolemia is a major risk factor for ischemic heart disease including acute myocardial infarction. However, long-term effects of hypercholesterolemia in a rodent myocardial ischemia-reperfusion injury model are unknown. Therefore, the effects of diet-induced hypercholesterolemia on cardiac function and remodeling were investigated up to eight weeks after myocardial ischemia-reperfusion (MI-R) injury which was induced in either normocholesterolemic (NC-MI) or hypercholesterolemic (HC-MI) APOE*3-Leiden mice. Methods: Left ventricular (LV) dimensions were serially assessed using parasternal long-axis echocardiography followed by LV pressure-volume measurements. Subsequently, infarct size and the inflammatory response were analyzed by histology and fluorescence-activated cell sorting (FACS) analysis. Results: Intrinsic LV function eight weeks after MI-R was significantly impaired in HC-MI compared to NC-MI mice as assessed by end-systolic pressure, dP/dtMAX, and -dP/dtMIN. Paradoxically, infarct size was significantly decreased in HC-MI compared to NC-MI mice, accompanied by an increased wall thickness. Hypercholesterolemia caused a pre-ischemic peripheral monocytosis, in particular of Ly-6Chi monocytes whereas accumulation of macrophages in the ischemic-reperfused myocardium of HC-MI mice was decreased. Conclusion: Diet-induced hypercholesterolemia caused impaired LV function eight weeks after MI-R injury despite a reduced post-ischemic infarct size. This was preceded by a pre-ischemic peripheral monocytosis, while there was a suppressed accumulation of inflammatory cells in the ischemic-reperfused myocardium after eight weeks. This experimental model using hypercholesterolemic APOE*3-Leiden mice exposed to MI-R seems suitable to study novel cardioprotective therapies in a more clinically relevant animal model. [ABSTRACT FROM AUTHOR]

The ability to appropriately mimic human disease is critical for using animal models as a tool for understanding virus pathogenesis. In the case of Nipah virus (NiV), infection of humans appears to occur either through inhalation, contact with or consumption of infected material. In two of these circumstances, respiratory or sinusoidal exposure represents a likely route of infection. In this study, intermediate-size aerosol particles (~7 μm) of NiV-Malaysia were used to mimic potential routes of exposure by focusing viral deposition in the upper respiratory tract. Our previous report showed this route of exposure extended the disease course and a single animal survived the infection. Here, analysis of the peripheral immune response found minimal evidence of systemic inflammation and depletion of B cells during acute disease. However, the animal that survived infection developed an early IgM response with rapid development of neutralizing antibodies that likely afforded protection. The increase in NiV-specific antibodies correlated with an expansion of the B cell population in the survivor. Cell-mediated immunity was not clearly apparent in animals that succumbed during the acute phase of disease. However, CD4+ and CD8+ effector memory cells increased in the survivor with correlating increases in cytokines and chemokines associated with cell-mediated immunity. Interestingly, kinetic changes of the CD4+ and CD8bright T cell populations over the course of acute disease were opposite from animals that succumbed to infection. In addition, increases in NK cells and basophils during convalescence of the surviving animal were also evident, with viral antigen found in NK cells. These data suggest that a systemic inflammatory response and “cytokine storm” are not major contributors to NiV-Malaysia pathogenesis in the AGM model using this exposure route. Further, these data demonstrate that regulation of cell-mediated immunity, in addition to rapid production of NiV specific antibodies, may be critical for surviving NiV infection. [ABSTRACT FROM AUTHOR]

Cancer cells harness immune checkpoints such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1) and indoleamine 2,3-dioxygenase 1 (IDO1) to evade immune control. Checkpoint inhibitors have demonstrated durable anti-tumor efficacy in human and preclinical models. Liver toxicity is one of the common immune-related adverse events associated with checkpoint inhibitors (CPIs) and its frequency and severity often increase significantly during CPI combination therapies. We aim to develop a mouse model to elucidate the immune mechanisms of CPI-associated liver toxicity. Co-administration of CTLA-4 blocking antibody, 9D9, and/or an IDO1 inhibitor, epacadostat in wild-type and PD-1-/- mice (to simulate the effect of PD1 blockade) synergistically induced liver injury and immune cell infiltration. Infiltrated cells were primarily composed of CD8+ T cells and positively associated with hepatocyte necrosis. Strikingly, sites of hepatocyte necrosis were frequently surrounded by clusters of mononuclear immune cells. CPI treatments resulted in increased expression of genes associated with hepatocyte cell death, leukocyte migration and T cell activation in the liver. In conclusion, blockade of immune checkpoints PD-1, CTLA-4, and IDO1 act synergistically to enhance T cell infiltration and activity in the liver, leading to hepatocyte death. [ABSTRACT FROM AUTHOR]

Opisthorchiasis caused by food-borne trematode Opisthorchis felineus is a substantial public health problem, with 17 million persons infected worldwide. This chronic disease is associated with hepatobiliary inflammation, cholangiocyte dysplasia, cholangiofibrosis, intraepithelial neoplasia, and even cholangiocarcinoma among chronically infected individuals. To provide first insights into the mechanism by which O. felineus infection causes precancerous liver lesions, we investigated the level of oxidative stress (lipid peroxidation byproducts and 8-hydroxy-2′-deoxyguanosine) as well as the time course profiles of chronic inflammation and fibrogenesis markers in the dynamics of opisthorchiasis from 1 month to 1.5 years postinfection in an experimental model based on golden hamsters Mesocricetus auratus. For the first time, we showed that O. felineus infection provokes time-dependent accumulation of oxidative hepatobiliary lesions in the injured liver of hamsters. In particular, over the course of infection, lipid peroxidation byproducts 4-hydroxynonenal and malondialdehyde were upregulated; these changes in general correlate with the dynamics of hepatic histopathological changes. We detected macrophages with various immunophenotypes and elevated levels of CD68, COX2, and CD163 in the O. felineus–infected animals. Meanwhile, there was direct time-dependent elevation of TNF-α (R = 0.79; p < 0.001) and CD163 protein levels (R = 0.58; p = 0.022). We also provide quantitative data about epithelial hyperplasia marker CK7 and a marker of myofibroblast activation (α smooth muscle actin). Our present data provide first insights into the histopathological mechanism by which O. felineus infection causes liver injuries. These findings support the inclusion of O. felineus in Group 1 of biological carcinogens. [ABSTRACT FROM AUTHOR]

Silicone is used widely for tissue augmentation in humans. However, late complications, such as delayed inflammation and capsular contracture, remain uncharacterized, despite their importance. In the present study, we aimed to determine whether mechanical and thermal damage induce capsular inflammation around a foreign body, and elucidate the biological mechanism underlying this phenomenon. We injected silicone into the subcutaneous layer of the skin of New Zealand white rabbits. The rabbits were divided into two groups: the control group received no treatment; in the experimental group, external force was applied near the injection silicone using high-intensity focused ultrasound (HIFU). Tissues near the injected silicone were harvested from both groups on Days 4, 7, and 30 after HIFU treatment for comparative analysis. Visual and histological examinations showed clearly increased inflammation in the experimental group compared with that in the control group. Furthermore, capsular tissue from the experimental group displayed markedly increased collagen production. Immunofluorescence revealed marked activation of macrophages in the early stages of inflammation (Days 4 and 7 after HIFU treatment), which decreased on Day 30. Assessment of cytokine activation showed significantly increased expression of heat shock protein (HSP)27, HSP60, HSP70, toll-like receptor (TLR)2, TLR4, and interleukin-8 in the experimental group. The expression of transforming growth factor-β1 did not increase significantly in the experimental group. In conclusion, damage to tissues around the injected silicone induced capsular inflammation. Macrophages and damage-associated molecular pattern molecules were involved in the early stages of inflammation. HSP release activated TLRs, which subsequently activated innate immunity and induced the inflammatory response. [ABSTRACT FROM AUTHOR]

Kaposi’s sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) belongs to the subfamily of Gammaherpesvirinae and is the etiological agent of Kaposi’s sarcoma as well as of two lymphoproliferative diseases: primary effusion lymphoma and multicentric Castleman disease. The KSHV life cycle is divided into a latent and a lytic phase and is highly regulated by viral immunomodulatory proteins which control the host antiviral immune response. Among them is a group of proteins with homology to cellular interferon regulatory factors, the viral interferon regulatory factors 1–4. The KSHV vIRFs are known as inhibitors of cellular interferon signaling and are involved in different oncogenic pathways. Here we characterized the role of the second vIRF protein, vIRF2, during the KSHV life cycle. We found the vIRF2 protein to be expressed in different KSHV positive cells with early lytic kinetics. Importantly, we observed that vIRF2 suppresses the expression of viral early lytic genes in both newly infected and reactivated persistently infected endothelial cells. This vIRF2-dependent regulation of the KSHV life cycle might involve the increased expression of cellular interferon-induced genes such as the IFIT proteins 1, 2 and 3, which antagonize the expression of early KSHV lytic proteins. Our findings suggest a model in which the viral protein vIRF2 allows KSHV to harness an IFN-dependent pathway to regulate KSHV early gene expression. [ABSTRACT FROM AUTHOR]

Effective vaccine design relies on accurate knowledge of protection against a pathogen, so as to be able to induce relevant and effective protective responses against it. An ideal Human Immunodeficiency virus (HIV) vaccine should induce humoral as well as cellular immune responses to prevent initial infection of host cells or limit early events of viral dissemination. A Phase I HIV-1 prophylactic vaccine trial sponsored by the International AIDS Vaccine Initiative (IAVI) was conducted in India in 2009.The trial tested a HIV-1 subtype C vaccine in a prime-boost regimen, comprising of a DNA prime (ADVAX) and Modified Vaccine Ankara (MVA) (TBC-M4) boost. The trial reported that the vaccine regimen was safe, well tolerated, and resulted in enhancement of HIV-specific immune responses. However, preliminary immunological studies were limited to vaccine-induced IFN-γ responses against the Env and Gag peptides. The present study is a retrospective study to characterize in detail the nature of the vaccine-induced cell mediated immune responses among volunteers, using Peripheral Blood Mononuclear Cells (PBMC) that were archived during the trial. ELISpot was used to measure IFN-γ responses and polyfunctional T cells were analyzed by intracellular multicolor flow cytometry. It was observed that DNA priming and MVA boosting induced Env and Gag specific bi-functional and multi-functional CD4+ and CD8+ T cells expressing IFN-γ, TNF-α and IL-2. The heterologous prime-boost regimen appeared to be slightly superior to the homologous prime-boost regimen in inducing favorable cell mediated immune responses. These results suggest that an in-depth analysis of vaccine-induced cellular immune response can aid in the identification of correlates of an effective immunogenic response, and inform future design of HIV vaccines. [ABSTRACT FROM AUTHOR]

Background: The application of polymeric materials in medical industry has grown drastically in the last two decades due to their various advantages compared to existing materials. The present research work emphases on the sol-gel technique to formulate the polymethyl methyl acrylate/polystyrene/silica composite membrane. Methods: The characteristic of the composite was investigated through modern state art of instrumentation. Results: The functional groups attached to the polymer was absorbed by FTIR. The FTIR spectrum confirm that the blend was mixed thoroughly and the formation of unite intimately between the polymers. The membranes were observed by SEM for its surface homogeneity which depends upon the composition of the two blending polymers. The captured SEM images showed the formation of microcracks on the surface, which was evidently controlled by varying the constituent polymer ratios. The prepared blend membranes with 2:1 ratio of PMMA/PS/Si displayed higher water uptake compared to other blended membranes. The composite membranes had good hydroxyl apatite growth in SBF solution. Furthermore, the in vitro cytotoxicity studies carried out by MTT method, using RAW macrophage cells showed that all the samples exhibited excellent cell viability. Conclusion: The inflammatory response of composite with equal concentration of PMMA-PS were performed and observed no inflammation in comparison with control and other tested concentrations. [ABSTRACT FROM AUTHOR]

This article describes the procedures used to isolate pure B-cell populations from whole blood using various Miltenyi magnetic-activated cell sorting (MACS) bead Isolation kits. Such populations are vital for studies investigating the functional capacity of B-cells, as the presence of other cell types may have indirect effects on B-cell function through cell-cell interactions or by secretion of several soluble molecules. B-cells can be isolated by two main approaches: 1) Negative selection—in which B-cells remain "untouched" in their native state; this is advantageous as it is likely that B-cells remain functionally unaltered by this process. 2) Positive selection–in which B-cells are labelled and actively removed from the sample. We used three Negative B-cell isolation kits as well as the Positive B-cell isolation kit from Miltenyi and compared the purity of each of the resulting B-cells fractions. Contamination of isolated B-cell fractions with platelets was the conclusive finding for all of the isolation techniques tested. These results illustrate the inefficiency of current available MACS B-cell isolation kits to produce pure B-cell populations, from which concrete findings can be made. As such we suggest cell sorting as the preferred method for isolating pure B-cells to be used for downstream functional assays. [ABSTRACT FROM AUTHOR]

The T-cell (TCR) repertoire relies on the diversity of receptors composed of two chains, called α and β, to recognize pathogens. Using results of high throughput sequencing and computational chain-pairing experiments of human TCR repertoires, we quantitively characterize the αβ generation process. We estimate the probabilities of a rescue recombination of the β chain on the second chromosome upon failure or success on the first chromosome. Unlike β chains, α chains recombine simultaneously on both chromosomes, resulting in correlated statistics of the two genes which we predict using a mechanistic model. We find that ∼35% of cells express both α chains. Altogether, our statistical analysis gives a complete quantitative mechanistic picture that results in the observed correlations in the generative process. We learn that the probability to generate any TCRαβ is lower than 10−12 and estimate the generation diversity and sharing properties of the αβ TCR repertoire. [ABSTRACT FROM AUTHOR]

Molecular mimicry is the formation of specific molecules by microbial pathogens to avoid recognition and attack by the immune system of the host. Several pathogenic Ascomycota and Zygomycota show such a behaviour by utilizing human complement factor H to hide in the blood stream. We call this type of mimicry molecular crypsis. Such a crypsis can reach a point where the immune system can no longer clearly distinguish between self and non-self cells. Thus, a trade-off between attacking disguised pathogens and erroneously attacking host cells has to be made. Based on signalling theory and protein-interaction modelling, we here present a mathematical model of molecular crypsis of pathogenic fungi using the example of Candida albicans. We tackle the question whether perfect crypsis is feasible, which would imply that protection of human cells by complement factors would be useless. The model identifies pathogen abundance relative to host cell abundance as the predominant factor influencing successful or unsuccessful molecular crypsis. If pathogen cells gain a (locally) quantitative advantage over host cells, even autoreactivity may occur. Our new model enables insights into the mechanisms of candidiasis-induced sepsis and complement-associated autoimmune diseases. [ABSTRACT FROM AUTHOR]

Neutrophils display rapid and potent innate immune responses in various diseases. Tumor-associated neutrophils (TANs) however either induce or overcome immunosuppressive functions of the tumor microenvironment through complex tumor-stroma crosstalk. We developed a mathematical model to address the question of how phenotypic alterations between tumor suppressive N1 TANS, and tumor promoting N2 TANs affect nonlinear tumor growth in a complex tumor microenvironment. The model provides a visual display of the complex behavior of populations of TANs and tumors in response to various TGF-β and IFN-β stimuli. In addition, the effect of anti-tumor drug administration is incorporated in the model in an effort to achieve optimal anti-tumor efficacy. The simulation results from the mathematical model were in good agreement with experimental data. We found that the N2-to-N1 ratio (N21R) index is positively correlated with aggressive tumor growth, suggesting that this may be a good prognostic factor. We also found that the antitumor efficacy increases when the relative ratio (Dap) of delayed apoptotic cell death of N1 and N2 TANs is either very small or relatively large, providing a basis for therapeutically targeting prometastatic N2 TANs. [ABSTRACT FROM AUTHOR]

Adhesion formation that occurred after alkali-induced injury of the cecum was used as a novel adhesion model in rats, and it was compared with that of a common adhesion model after abrading the cecum. Using the novel adhesion model, inhibition of adhesion formation by a chymase inhibitor, Suc-Val-Pro-PheP(OPh)2, and by sodium hyaluronate/carboxymethylcellulose (Seprafilm) was evaluated, and their mechanisms were assessed. The degree of adhesion formation was more severe and more stable in the alkali-induced injury model than in the abrasion-induced injury model. Both the chymase inhibitor and Seprafilm showed significant attenuation of the degree of adhesion 14 days after alkali-induced injury. Chymase activity in the cecum was significantly increased after alkali-induced injury, but it was significantly attenuated by the chymase inhibitor and Seprafilm. Myeloperoxidase and transforming-growth factor (TGF)-β levels were significantly increased after alkali-induced injury, but they were attenuated by both the chymase inhibitor and Seprafilm. At the level of the adhesions, the numbers of both chymase-positive cells and TGF-β-positive cells were significantly increased, but their numbers were reduced by the chymase inhibitor and Seprafilm. In conclusion, a chymase inhibitor attenuated the degree of adhesions to the same degree as Seprafilm in a novel peritoneal adhesion model that was more severe and more stable than the common adhesion model, and not only the chymase inhibitor, but also Seprafilm reduced the chymase increase at the adhesions. [ABSTRACT FROM AUTHOR]

Deep subcortical lesions (DSCL) of the brain, are present in ~60% of the ageing population, and are linked to cognitive decline and depression. DSCL are associated with demyelination, blood brain barrier (BBB) dysfunction, and microgliosis. Microglia are the main immune cell of the brain. Under physiological conditions microglia have a ramified morphology, and react to pathology with a change to a more rounded morphology as well as showing protein expression alterations. This study builds on previous characterisations of DSCL and radiologically ‘normal-appearing’ white matter (NAWM) by performing a detailed characterisation of a range of microglial markers in addition to markers of vascular integrity. The Cognitive Function and Ageing Study (CFAS) provided control white matter (WM), NAWM and DSCL human post mortem tissue for immunohistochemistry using microglial markers (Iba-1, CD68 and MHCII), a vascular basement membrane marker (collagen IV) and markers of BBB integrity (fibrinogen and aquaporin 4). The immunoreactive profile of CD68 increased in a stepwise manner from control WM to NAWM to DSCL. This correlated with a shift from small, ramified cells, to larger, more rounded microglia. While there was greater Iba-1 immunoreactivity in NAWM compared to controls, in DSCL, Iba-1 levels were reduced to control levels. A prominent feature of these DSCL was a population of Iba-1-/CD68+ microglia. There were increases in collagen IV, but no change in BBB integrity. Overall the study shows significant differences in the immunoreactive profile of microglial markers. Whether this is a cause or effect of lesion development remains to be elucidated. Identifying microglia subpopulations based on their morphology and molecular markers may ultimately help decipher their function and role in neurodegeneration. Furthermore, this study demonstrates that Iba-1 is not a pan-microglial marker, and that a combination of several microglial markers is required to fully characterise the microglial phenotype. [ABSTRACT FROM AUTHOR]

Non-coding RNAs have important roles in regulating physiology, including immunity. Here, we performed transcriptome profiling of immune-responsive genes in Drosophila melanogaster during a Gram-positive bacterial infection, concentrating on long non-coding RNA (lncRNA) genes. The gene most highly induced by a Micrococcus luteus infection was CR44404, named Induced by Infection (lincRNA-IBIN). lincRNA-IBIN is induced by both Gram-positive and Gram-negative bacteria in Drosophila adults and parasitoid wasp Leptopilina boulardi in Drosophila larvae, as well as by the activation of the Toll or the Imd pathway in unchallenged flies. We show that upon infection, lincRNA-IBIN is expressed in the fat body, in hemocytes and in the gut, and its expression is regulated by NF-κB signaling and the chromatin modeling brahma complex. In the fat body, overexpression of lincRNA-IBIN affected the expression of Toll pathway -mediated genes. Notably, overexpression of lincRNA-IBIN in unchallenged flies elevated sugar levels in the hemolymph by enhancing the expression of genes important for glucose retrieval. These data show that lncRNA genes play a role in Drosophila immunity and indicate that lincRNA-IBIN acts as a link between innate immune responses and metabolism. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMP), part of the innate immune system, are well studied for their ability to kill pathogenic microorganisms. However, many also possess important immunomodulatory effects, and this area has potential for the development of novel therapies to supplement traditional methods such as the use of antibiotics. Here, we characterise the microbicidal and immunomodulatory potential of the proline-rich bovine AMP, Bactenecin 5 (Bac5). We demonstrate broad antimicrobial activity, including against some mycobacterial species, which are important pathogens of fish, cattle and humans. Bac5 is able to activate macrophage-like THP-1 cells and can synergistically trigger the upregulation of tnf-α when co-stimulated with M. marinum. Furthermore, Bac5 sensitises A549 epithelial cells to stimulation with TNF-α. For the first time, we characterise the activity of Bac5 in vivo, and show it to be a potent chemokine for macrophages in the zebrafish (Danio rerio) embryo model of infection. Bac5 also supports the early recruitment of neutrophils in the presence of M. marinum. In the absence of host adaptive immunity, exogenous injected Bac5 is able to slow, although not prevent, infection of zebrafish with M. marinum. [ABSTRACT FROM AUTHOR]

Rationale: HSP90B1, also known as gp96, is a chaperone for multiple Toll-like receptors (TLRs) and is necessary for TLR-mediated inflammatory responses in murine myeloid cells. The molecule is also expressed in T-cells though its specific role is unknown. We hypothesized that human HSP90B1 regulates monocyte and T-cell responses to Mycobacterium tuberculosis (Mtb) and bacilli Calmette-Guerin (BCG) and that its variants are associated with susceptibility to TB disease. Methods: We screened 17 haplotype-tagging SNPs in the HSP90B1 gene region for association with BCG-induced T-cell cytokine responses using both an ex-vivo whole blood assay (N = 295) and an intracellular cytokine staining assay (N = 180) on samples collected 10 weeks after birth. Using a case-control study design, we evaluated the same SNPs for association with TB disease in a South African pediatric cohort (N = 217 cases, 604 controls). A subset of these SNPs was evaluated for association with HSP90B1 expression in human monocytes, monocyte-derived dendritic cells, and T-cells using RT-PCR. Lastly, we used CRISPR/Cas9 gene editing to knock down HSP90B1 expression in a human monocyte cell line (U937). Knockdown and control cell lines were tested for TLR surface expression and control of Mtb replication. Results: We identified three SNPs, rs10507172, rs10507173 and rs1920413, that were associated with BCG-induced IL-2 secretion (p = 0.017 for rs10507172 and p = 0.03 for rs10507173 and rs1920413, Mann-Whitney, dominant model). SNPs rs10507172 and rs10507173 were associated with TB disease in an unadjusted analysis (p = 0.036 and 0.025, respectively, dominant model) that strengthened with sensitivity analysis of the definite TB cases, which included only those patients with microbiologically confirmed Mtb (p = 0.007 and 0.012, respectively). Knockdowns of HSP90B1 in monocyte cell lines with CRISPR did not alter TLR2 surface expression nor influence Mtb replication relative to controls. Conclusion: Among infants, an HSP90B1 gene-region variant is associated with BCG-induced IL-2 production and may be associated with protection from TB disease. HSP90B1 knockdown in human monocyte-like cell lines did not influence TLR2 surface localization nor Mtb replication. Together, these data suggest that HSP90B1 regulates T-cell, but not monocyte, responses to mycobacteria in humans. [ABSTRACT FROM AUTHOR]

CCR5 plays immune functions and is the coreceptor for R5 HIV-1 strains. It exists in diverse conformations and oligomerization states. We interrogated the significance of the CCR5 structural diversity on HIV-1 infection. We show that envelope glycoproteins (gp120s) from different HIV-1 strains exhibit divergent binding levels to CCR5 on cell lines and primary cells, but not to CD4 or the CD4i monoclonal antibody E51. This owed to differential binding of the gp120s to different CCR5 populations, which exist in varying quantities at the cell surface and are differentially expressed between different cell types. Some, but not all, of these populations are antigenically distinct conformations of the coreceptor. The different binding levels of gp120s also correspond to differences in their capacity to bind CCR5 dimers/oligomers. Mutating the CCR5 dimerization interface changed conformation of the CCR5 homodimers and modulated differentially the binding of distinct gp120s. Env-pseudotyped viruses also use particular CCR5 conformations for entry, which may differ between different viruses and represent a subset of those binding gp120s. In particular, even if gp120s can bind both CCR5 monomers and oligomers, impairment of CCR5 oligomerization improved viral entry, suggesting that HIV-1 prefers monomers for entry. From a functional standpoint, we illustrate that the nature of the CCR5 molecules to which gp120/HIV-1 binds shapes sensitivity to inhibition by CCR5 ligands and cellular tropism. Differences exist in the CCR5 populations between T-cells and macrophages, and this is associated with differential capacity to bind gp120s and to support viral entry. In macrophages, CCR5 structural plasticity is critical for entry of blood-derived R5 isolates, which, in contrast to prototypical M-tropic strains from brain tissues, cannot benefit from enhanced affinity for CD4. Collectively, our results support a role for CCR5 heterogeneity in diversifying the phenotypic properties of HIV-1 isolates and provide new clues for development of CCR5-targeting drugs. [ABSTRACT FROM AUTHOR]

Sequencing of the T cell receptor repertoire is a powerful tool for deeper study of immune response, but the unique structure of this type of data makes its meaningful quantification challenging. We introduce a new method, the Gamma-GPD spliced threshold model, to address this difficulty. This biologically interpretable model captures the distribution of the TCR repertoire, demonstrates stability across varying sequencing depths, and permits comparative analysis across any number of sampled individuals. We apply our method to several datasets and obtain insights regarding the differentiating features in the T cell receptor repertoire among sampled individuals across conditions. We have implemented our method in the open-source R package powerTCR. [ABSTRACT FROM AUTHOR]

Background: Peritumoral inflammation—a response mainly involving polimorphonuclear neutrophils—has traditionally been thought protumoral in its effects. In recent years, however, a number of studies have indicated that it may play an important antitumoral role. This discrepancy has been difficult to explain. Methods and findings: This work describes a tool for simulating tumor growth that obeys the universal model of tumor growth dynamics, and shows through its use that low intensity peritumoral inflammation exerts a protumoral effect, while high intensity inflammation exerts a potent antitumoral effect. Indeed, the simulation results obtained indicate that a sufficiently strong antitumoral effect can reverse tumor growth, as has been suggested several times in the clinical literature. Conclusions: The present result indicate that an ‘immunological threshold’ must exist, marking the boundary between states in which peritumoral inflammation is either harmful or beneficial. These findings lend support to the idea that stimulating intense peritumoral inflammation could be used as a treatment against solid tumors. [ABSTRACT FROM AUTHOR]

Data from SIV-infected macaques indicate that virus-specific cytotoxic T lymphocytes (CTL) are mostly present in the extrafollicular (EF) compartment of the lymphoid tissue, with reduced homing to the follicular (F) site. This contributes to the majority of the virus being present in the follicle and represents a barrier to virus control. Using mathematical models, we investigate these dynamics. Two models are analyzed. The first assumes that CTL can only become stimulated and expand in the extrafollicular compartment, with migration accounting for the presence of CTL in the follicle. In the second model, follicular CTL can also undergo antigen-induced expansion. Consistent with experimental data, both models predict increased virus compartmentalization in the presence of stronger CTL responses and lower virus loads, and a more pronounced rise of extrafollicular compared to follicular virus during CD8 cell depletion experiments. The models, however, differ in other aspects. The follicular expansion model results in dynamics that promote the clearance of productive infection in the extrafollicular site, with any productively infected cells found being the result of immigration from the follicle. This is not observed in the model without follicular CTL expansion. The models further predict different consequences of introducing engineered, follicular-homing CTL, which has been proposed as a therapeutic means to improve virus control. Without follicular CTL expansion, this is predicted to result in a reduction of virus load in both compartments. The follicular CTL expansion model, however, makes the counter-intuitive prediction that addition of F-homing CTL not only results in a reduction of follicular virus load, but also in an increase in extrafollicular virus replication. These predictions remain to be experimentally tested, which will be relevant for distinguishing between models and for understanding how therapeutic introduction of F-homing CTL might impact the overall dynamics of the infection. [ABSTRACT FROM AUTHOR]

Purpose: As atherosclerotic plaque ruptures are the primary cause of ischaemic events, their preventive identification by imaging remains a clinical challenge. Matrix metalloproteinases (MMP) are involved in plaque progression and destabilisation and are therefore promising targets to characterize rupture-prone unstable plaques. This study aims at evaluating MMP imaging to discriminate unstable from stable plaque phenotypes. Methods: ApoE deficient mice (ApoE-/-) on a high cholesterol diet underwent implantation of a tapered cuff around the right common carotid artery (CCA) inducing a highly inflamed atherosclerotic plaque upstream (US) and a more stable plaque phenotype downstream (DS) of the cuff. 8 weeks after surgery, the MMP inhibitor-based photoprobe Cy5.5-AF443 was administered i.v. 3h prior to in situ and ex vivo fluorescence reflectance imaging of the CCAs. Thereafter, CCAs were analysed regarding plaque size, presence of macrophages, and MMP-2 and MMP-9 concentrations by immunohistochemistry and ELISA. Results: We found a significantly higher uptake of Cy5.5-AF443 in US as compared to DS plaques in situ (1.29 vs. 1.06 plaque-to-background ratio; p<0.001), which was confirmed by ex vivo measurements. Immunohistochemistry revealed a higher presence of macrophages, MMP-2 and MMP-9 in US compared to DS plaques. Accordingly, MMP-2 concentrations were significantly higher in US plaques (47.2±7.6 vs. 29.6±4.6 ng/mg; p<0.05). Conclusions: In the ApoE-/- cuff model MMP-2 and MMP-9 activities are significantly higher in upstream low shear stress-induced unstable atherosclerotic plaques as compared to downstream more stable plaque phenotypes. MMP inhibitor-based fluorescence molecular imaging allows visualization of these differences in shear stress-induced atherosclerosis. [ABSTRACT FROM AUTHOR]

Aims: Endothelial activation is involved in many chronic inflammatory diseases, such as atherosclerosis, and is often initiated by cytokines. Oncostatin M (OSM) is a relatively unknown cytokine that has been suggested to play a role in both endothelial activation and atherosclerosis. We comprehensively investigated the effect of OSM on endothelial cell activation from different vascular beds and in APOE*3Leiden.CETP mice. Methods and results: Human umbilical vein endothelial cells, human aortic endothelial cells and human microvascular endothelial cells cultured in the presence of OSM express elevated MCP-1, IL-6 and ICAM-1 mRNA levels. Human umbilical vein endothelial cells and human aortic endothelial cells additionally expressed increased VCAM-1 and E-selectin mRNA levels. Moreover, ICAM-1 membrane expression is increased as well as MCP-1, IL-6 and E-selectin protein release. A marked increase was observed in STAT1 and STAT3 phosphorylation indicating that the JAK/STAT pathway is involved in OSM signaling. OSM signals through the LIF receptor alfa (LIFR) and the OSM receptor (OSMR). siRNA knockdown of the LIFR and the OSMR revealed that simultaneous knockdown is necessary to significantly reduce MCP-1 and IL-6 secretion, VCAM-1 and E-selectin shedding and STAT1 and STAT3 phosphorylation after OSM stimulation. Moreover, OSM administration to APOE*3Leiden.CETP mice enhances plasma E-selectin levels and increases ICAM-1 expression and monocyte adhesion in the aortic root area. Furthermore, Il-6 mRNA expression was elevated in the aorta of OSM treated mice. Conclusion: OSM induces endothelial activation in vitro in endothelial cells from different vascular beds through activation of the JAK/STAT cascade and in vivo in APOE*3Leiden.CETP mice. Since endothelial activation is an initial step in atherosclerosis development, OSM may play a role in the initiation of atherosclerotic lesion formation. [ABSTRACT FROM AUTHOR]

Countless neurodegenerative diseases are associated with perverse multiple targets of cyclic nucleotide signalling, hastening neuronal death. Cilostazol, a phosphodiesterase-III inhibitor, exerts neuroprotective effects against sundry models of neurotoxicity, however, its role against Huntington’s disease (HD) has not yet been tackled. Hence, its modulatory effect on several signalling pathways using the 3-nitropropionic acid (3-NP) model was conducted. Animals were injected with 3-NP (10 mg/kg/day, i.p) for two successive weeks with or without the administration of cilostazol (100 mg/kg/day, p.o.). Contrary to the 3-NP effects, cilostazol largely preserved striatal dopaminergic neurons, improved motor coordination, and enhanced the immunohistochemical reaction of tyrosine hydroxylase enzyme. The anti-inflammatory effect of cilostazol was documented by the pronounced reduction of the toll like receptor-4 (TLR-4) protein expression and the inflammatory cytokine IL-6, but with a marked elevation in IL-10 striatal contents. As a consequence, cilostazol reduced IL-6 downstream signal, where it promoted the level of suppressor of cytokine signalling 3 (SOCS3), while abated the phosphorylation of Janus Kinase 2 (JAK-2) and Signal transducers and activators of transcription 3 (STAT-3). Phosphorylation of the protein kinase B/glycogen synthase kinase-3β/cAMP response element binding protein (Akt/GSK-3β/CREB) cue is another signalling pathway that was modulated by cilostazol to further signify its anti-inflammatory and antiapoptotic capacities. The latter was associated with a reduction in the caspase-3 expression assessed by immunohistochemical assay. In conclusion the present study provided a new insight into the possible mechanisms by which cilostazol possesses neuroprotective properties. These intersecting mechanisms involve the interference between TLR-4, IL-6-IL-10/JAK-2/STAT-3/SOCS-3, and Akt/GSK-3β/CREB signalling pathways. [ABSTRACT FROM AUTHOR]

Innate immunity is an ancient and conserved defense system that provides an early effective response against invaders. Many immune genes of Anopheles mosquitoes have been implicated in defense against a variety of pathogens, including plasmodia. Nevertheless, only recent work identified some immune genes of Anopheles aquasalis mosquitoes upon P. vivax infection. Among these was a GATA transcription factor gene, which is described here. This is an ortholog of GATA factor Serpent genes described in Drosophila melanogaster and Anopheles gambiae. Gene expression analyses showed an increase of GATA-Serpent mRNA in P. vivax-infected A. aquasalis and functional RNAi experiments identified this transcription factor as an important immune gene of A. aquasalis against both bacteria and P. vivax. Besides, we were able to identify an effect of GATA-Serpent knockdown on A. aquasalis hemocyte proliferation and differentiation. These findings expand our understanding of the poorly studied A. aquasalis-P. vivax interactions and uncover GATA-Serpent as a key player of the mosquito innate immune response. [ABSTRACT FROM AUTHOR]

Background: The addition of plasmid cytokine adjuvants, electroporation, and live attenuated viral vectors may further optimize immune responses to DNA vaccines in heterologous prime-boost combinations. The objective of this study was to test the safety and tolerability of a novel prime-boost vaccine regimen incorporating these strategies with different doses of IL-12 plasmid DNA adjuvant. Methods: In a phase 1 study, 88 participants received an HIV-1 multiantigen (gag/pol, env, nef/tat/vif) DNA vaccine (HIV-MAG, 3000 μg) co-administered with IL-12 plasmid DNA adjuvant at 0, 250, 1000, or 1500 μg (N = 22/group) given intramuscularly with electroporation (Ichor TriGrid™ Delivery System device) at 0, 1 and 3 months; followed by attenuated recombinant vesicular stomatitis virus, serotype Indiana, expressing HIV-1 Gag (VSV-Gag), 3.4 ⊆ 107 plaque-forming units (PFU), at 6 months; 12 others received placebo. Injections were in both deltoids at each timepoint. Participants were monitored for safety and tolerability for 15 months. Results: The dose of IL-12 pDNA did not increase pain scores, reactogenicity, or adverse events with the co-administered DNA vaccine, or following the VSV-Gag boost. Injection site pain and reactogenicity were common with intramuscular injections with electroporation, but acceptable to most participants. VSV-Gag vaccine often caused systemic reactogenicity symptoms, including a viral syndrome (in 41%) of fever, chills, malaise/fatigue, myalgia, and headache; and decreased lymphocyte counts 1 day after vaccination. Conclusions: HIV-MAG DNA vaccine given by intramuscular injection with electroporation was safe at all doses of IL-12 pDNA. The VSV-Gag vaccine at this dose was associated with fever and viral symptoms in some participants, but the vaccine regimens were safe and generally well-tolerated. Trial registration: Clinical Trials.gov . [ABSTRACT FROM AUTHOR]

We had previously reported that exchange protein for cAMP 1 (Epac1) reduced inflammatory mediators in the retina of mice and in retinal endothelial cells (REC). Since ischemia can induce retinal damage potentially through activation of inflammatory cascades, we hypothesized that Epac1 would protect the retina against neuronal and vascular damage after exposure to ischemia/reperfusion (I/R). We used Epac1 floxed and endothelial cell specific Epac1 knockout mice for this work. We exposed them to ischemia for 90 minutes followed by reperfusion. One day after I/R, some mice were used for fluorescein angiography imaging or Evan’s blue measurements of permeability. Mice were sacrificed at 2 days for neuronal measurements and at 10 days for measurements of degenerate capillaries. Data show increased leakage in the Epac1 Cre-Lox (Epac1 EC-KO) mice exposed to I/R when compared to Epac1 floxed mice with the same treatment. I/R also increased numbers of degenerate capillaries and cell loss in all retinal layers of Epac1 EC-KO mice. Retinal thickness was reduced more significantly in the Epac1 EC-KO mice compared to Epac1 floxed mice after I/R. Taken together, the data suggest that Epac1 is protective against both neuronal and vascular damage to the retina after exposure to I/R. [ABSTRACT FROM AUTHOR]

Growing evidence highlights the peripheral blood mononuclear cells (PBMCs) role and the chemokine involvement in the Alzheimer’s disease (AD) physiopathology. However, few data are available about the impact of AD PBMCs in the chemokine signature in a brain with AD phenotype. Therefore, this study analyzed the chemokine levels in a human blood brain barrier model. A human endothelial cell line from the immortalized cerebral microvascular endothelial cell line (hCMEC/D3) and a human glioblastoma U-87 MG cell line, both with no AD phenotype were used while PBMCs came from AD at mild or moderate stage and control patients. PBMCs from moderate AD patients decreased CCL2 and CCL5 levels in endothelial, and also CXCL10 in abluminal compartments and in PBMCs compared to PBMCs from mild AD patients. The CX3CL1 expression increased in endothelial and abluminal compartments with PBMCs from mild AD patients compared to controls. AD PBMCs can convert the chemokine signature towards that found in AD brain, targeting some chemokines as new biomarkers in AD. [ABSTRACT FROM AUTHOR]

Antibody dependent enhancement (ADE) has most often been associated with dengue virus (DENV). Studies using leukemia cell lines suggest that DENV specific antibodies can enhance Zika virus (ZIKV) infectivity, and vice versa. To examine the mechanisms of ADE of ZIKV infection in primary human cells, we assessed 40 serum samples obtained from convalescent DENV-1 or DENV-3 infected subjects. All sera tested exhibited high binding potency, while modest or none neutralization activities against ZIKV. Primary CD14+ monocytes, rather than B and T cells in peripheral blood mononuclear cells (PBMCs), were found to be the mediators of the enhancement of ZIKV infectivity by DENV immune sera. Monocyte-derived immature dendritic cells (DCs), but not mature DCs were highly permissive to ZIKV infection, whereas neither immature nor mature DCs could mediate enhanced ZIKV infection in the presence of DENV immune sera. In addition, antibody blocking of either FcγRI (CD64), or FcγRII (CD32), or FcγRIII (CD16) resulted in diminished ADE of ZIKV infection. Our findings provide an improved understanding of the pathogenesis of ZIKV infection, and inform rational vaccine design. [ABSTRACT FROM AUTHOR]

Background: Different inflammatory reactions have been observed in the polyp tissues of nonsmokers and smokers with chronic rhinosinusitis (CRS). E-prostanoid (EP) receptors play a role in the inflammatory processes. Cigarette smoke (CS) exposure regulates EP-receptor expression levels promoting inflammatory mediator release from various inflammatory cells. In this study, we characterize the EP-receptor expression profiles in the polyps of nonsmoking and smoking CRS patients to explore the possible role of CS in the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP). Methods: Polyp biopsies were obtained from 28 non-smoking and 21 smoking CRSwNP patients. Histopathological characteristics were observed under a light microscope. The prostaglandin E2 (PGE2), TNF-α, and IL-8 contents in polyp tissues were detected using enzyme-linked immunosorbent assay. Immunostaining was used to locate EP receptors in polyps. Messenger RNA and protein expression of EP receptors were examined using quantitative real-time polymerase chain reaction and Western blot, respectively. Results: More severe inflammatory reactions occurred in polyp tissues of smoking CRSwNP patients. The PGE2, TNF-α, and IL-8 in tissue homogenate levels were significantly higher in smoking CRSwNP patients than those in nonsmoking CRSwNP patients. Moreover, the distribution of each EP receptor subtype was similar in both groups. Compared with the EP-receptor expression in nonsmokers, messenger RNA and protein of EP2 and EP4 receptor were significantly down-expressed in smoking patients, but EP1 and EP3 receptors did not show significant differences. Conclusion: CS exposure downregulates the expression levels of EP2 and EP4 receptors and stimulates the production of PGE2 and the proinflammatory cytokine IL-8 and TNF-α in polyp tissues of CRS patients. The down-expressed EP2 and EP4 receptors might be associated with severe inflammatory reactions in smoking CRSwNP patients. [ABSTRACT FROM AUTHOR]

Congenital Cytomegalovirus infection (cCMV) is the leading infection in determining permanent long-term impairments (LTI), and its pathogenesis is largely unknown due to the complex interplay between viral, maternal, placental, and child factors. The cellular activity, considered to be the result of the response to exogenous and endogenous factors, is captured by the determination of gene expression profiles. In this study, we determined whole blood transcriptomes in relation to cCMV, CMV viral load and LTI development at 6 years of age by using RNA isolated from neonatal dried blood spots (DBS) stored at room temperature for 8 years. As DBS were assumed to mainly reflect the neonatal immune system, particular attention was given to the immune pathways using the global test. Additionally, differential expression of individual genes was performed using the voom/limma function packages. We demonstrated feasibility of RNA sequencing from archived neonatal DBS of children with cCMV, and non-infected controls, in relation to LTI and CMV viral load. Despite the lack of statistical power to detect individual genes differences, pathway analysis suggested the involvement of innate immune response with higher CMV viral loads, and of anti-inflammatory markers in infected children that did not develop LTI. Finally, the T cell exhaustion observed in infected neonates, in particular with higher viral load, did not correlate with LTI, therefore other mechanisms are likely to be involved in the long-term immune dysfunction. Despite these data demonstrate limitation in determining prognostic markers for LTI by means of transcriptome analysis, this exploratory study represents a first step in unraveling the pathogenesis of cCMV, and the aforementioned pathways certainly merit further evaluation. [ABSTRACT FROM AUTHOR]

Purpose: This study was to evaluate whether optic nerve damage occurs in eyes with adjacent chronic sinusitis. Methods: Data were collected from eighty-eight eyes of 46 chronic sinusitis patients and 93 eyes of 57 normal controls. Visual sensitivity using standard automated perimetry (SAP) and inner retinal thickness using optical coherence tomography (OCT) were measured. The Lund-Mackay system was used to quantify radiographic findings on the ostiomeatal unit CT scan with a numerical score representing the severity of sinusitis. Results: There was a significant positive correlation between the pattern standard deviation (dB) and Lund-Mackay score (P = 0.031). Nasal retinal nerve fiber layer (RNFL) thickness, average, minimum, superotemporal, superior, superonasal, and inferonasal ganglion cell-inner plexiform layer (GCIPL) thickness were negatively correlated significantly with Lund-Mackay score (all, P < 0.05). Eyes with grade 2 opacification of the posterior ethmoid sinus showed a significantly lower mean deviation (dB) and higher pattern standard deviation (dB) than those with clear respective sinuses (P = 0.007 and <0.001, respectively). Eyes with grades 1,2 and 3 opacification of the sphenoid sinus had a significantly less average RNFL thickness (P = 0.004, <0.001, and <0.001, respectively) and a significantly less average GCIPL thickness (P = 0.004, 0.003, and 0.003, respectively) than those with a clear sphenoid sinus. Conclusions: Structural and functional optic nerve changes were correlated with the severity of chronic sinusitis. Inflammation of the posterior ethmoid and sphenoid sinuses was associated with optic nerve changes to a greater extent than that of the other paranasal sinuses. [ABSTRACT FROM AUTHOR]

The field of dermal fillers is evolving rapidly and numerous products are currently on the market. Biodegradable polymers such as polycaprolactone (PCL) have been found to be compatible with several body tissues, and this makes them an ideal material for dermal filling purposes. Hollow PCL spheres were developed by the Council for Scientific and Industrial Research (CSIR) to serve both as an anchor point and a “tissue harbour” for cells. Particles were tested for cytotoxicity and cell adherence using mouse embryo fibroblasts (MEF). MEFs adhered to the particles and no significant toxic effects were observed based on morphology, cell growth, cell viability and cell cycle analysis, suggesting that the particles are suitable candidates for cell delivery systems in an in vivo setting. The objective of providing a “tissue harbour” was however not realized, as cells did not preferentially migrate into the ported particles. In vivo studies were conducted in BALB/c mice into whom particles were introduced at the level of the hypodermis. Mice injected with PCL particles (ported and non-ported; with or without MEFs) showed evidence of local inflammation and increased adipogenesis at the site of injection, as well as a systemic inflammatory response. These effects were also observed in mice that received apparently inert (polystyrene) particles. Ported PCL particles can therefore act as a cell delivery system and through their ability to induce adipogenesis, may also serve as a dermal bulking agent. [ABSTRACT FROM AUTHOR]

Differentiating early mycosis fungoides (MF) from inflammatory dermatitis is a challenge. We compare the differential expression profile of early-stage MF samples and benign inflammatory dermatoses using microRNA (miRNA) arrays. 114 miRNAs were found to be dysregulated between these entities. The seven most differentially expressed miRNAs between these two conditions were further analyzed using RT-PCR in two series comprising 38 samples of early MFs and 18 samples of inflammatory dermatitis. A series of 51 paraffin-embedded samples belonging to paired stages of 16 MF patients was also analyzed. MiRNAs 26a, 222, 181a and 146a were differentially expressed between tumoral and inflammatory conditions. Two of these miRNAs (miRNA-181a and miRNA-146a) were significantly deregulated between early and advanced MF stages. Bioinformatic analysis showed FOXP3 expression to be regulated by these miRNAs. Immunohistochemistry revealed the level of FOXP3 expression to be lower in tumoral MFs than in plaque lesions in paraffin-embedded tissue. A functional study confirmed that both miRNAs diminished FOXP3 expression when overexpressed in CTCL cells. The data presented here suggest that the analysis of a restricted number of miRNAs (26a, 222, 181a and 146a) could be sufficient to differentiate tumoral from reactive conditions. Moreover, these miRNAs seem to be involved in MF progression. [ABSTRACT FROM AUTHOR]