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Background: Increasing evidence supports the role of soluble inflammatory mediators in the pathogenesis of refractory temporal lobe epilepsy (TLE). Hippocampal sclerosis (HS) is a well-described pathohistological abnormality in TLE. The association of proinflammatory cytokines with epileptic disease profiles is well established; however, the potential significance of circulating interleukin 10 (IL-10), particularly in TLE-associated HS, is still poorly understood. Therefore, taking into consideration the neuroprotective and anticonvulsive effects of IL-10, we performed this study to examine the role of the plasma levels of IL-10 in patients with TLE with HS (TLE + HS), TLE without HS (TLE-HS) and with other types of epilepsy.Methods: This study included 270 patients with refractory epilepsy who were classified into four groups: i) 34 patients with TLE + HS, ii) 105 patients with TLE-HS, iii) 95 patients with extra-TLE (XLE) and iv) 36 patients with idiopathic generalized epilepsy (IGE). The plasma IL-10 levels were quantified using a commercially available enzyme-linked immunosorbent assay (ELISA).Results: IL-10 levels were significantly lower in TLE + HS than in TLE-HS (p = 0.013). In a subgroup of TLE-HS patients who had seizures 1 month before sampling, patients with seizures had significantly higher IL-10 levels than patients who were seizure-free (p = 0.039). Among a small group (n = 15) of non-refractory TLE-HS patients, IL-10 levels showed a moderate negative correlation with the duration of epilepsy (r = - 0.585, p = 0.023).Conclusions: This study demonstrated that chronically reduced levels of plasma IL-10 were associated with HS in TLE patients, suggesting that there was an inadequate systemic anti-inflammatory immune response. These results could provide new biological insights into the pathophysiology of HS in TLE. We also found that the production of IL-10 could be affected by the seizure frequency and declined concomitantly with increased disease durations. Therefore, the measurement of plasma IL-10 may have diagnostic value as a biomarker for stratifying TLE + HS from other epilepsy types or as a marker of disease progression towards a progressive form of epilepsy. [ABSTRACT FROM AUTHOR]

Mitogen-activated protein kinases ( MAPKs) include p38 MAPKs, c-Jun N-terminal kinases ( JNKs) and Extracellular signal-regulated kinases ( ERKs), and they regulate many cell processes, such as cell division, differentiation and release of inflammatory mediators. MAPK activity is controlled by mitogen-activated protein kinase phosphatases ( MKPs), a phosphatase family with 11 members. MKP-1 is the most studied member of MKP family, and it is one of the anti-inflammatory factors induced by glucocorticoids. Less is known about the other MAPK phosphatases although they hold a promise as anti-inflammatory drug targets. In this study, we investigated the effect of MKP-5 on MAPK phosphorylation and cytokine production in J774 mouse macrophages. We used MKP-5 si RNA and an MKP-5 inhibitor ( AS077234-4) to modulate MKP-5 function. We found that MKP-5 controlled p38 MAPK phosphorylation, but not that of JNK or ERK. In addition, the production of IL-6 and TNF was suppressed by MKP-5 in macrophages. Our results introduce a novel concept that compounds able to enhance MKP-5 expression and/or activity hold anti-inflammatory potential, because MKP-5 down-regulates the release of inflammatory mediators by controlling p38 MAPK activity. [ABSTRACT FROM AUTHOR]

Snd1 is an evolutionarily conserved RNA‐binding protein implicated in several regulatory processes in gene expression including activation of transcription, mRNA splicing, and microRNA decay. Here, we have investigated the outcome of Snd1 gene deletion in the mouse. The knockout mice are viable showing no gross abnormalities apart from decreased fertility, organ and body size, and decreased number of myeloid cells concomitant with decreased expression of granule protein genes. Deletion of Snd1 affected the expression of relatively small number of genes in spleen and liver. However, mRNA expression changes in the knockout mouse liver showed high similarity to expression profile in adaptation to hypoxia. MicroRNA expression in liver showed upregulation of the hypoxia‐induced microRNAs miR‐96 and ‐182. Similar to Snd1 deletion, mimics of miR‐96/182 enhanced hypoxia‐responsive reporter activity. To further elucidate the function of SND1, BioID biotin proximity ligation assay was performed in HEK‐293T cells to identify interacting proteins. Over 50% of the identified interactors were RNA‐binding proteins, including stress granule proteins. Taken together, our results show that in normal growth conditions, Snd1 is not a critical factor for mRNA transcription in the mouse, and describe a function for Snd1 in hypoxia adaptation through negatively regulating hypoxia‐related miRNAs and hypoxia‐induced transcription consistent with a role as stress response regulator. [ABSTRACT FROM AUTHOR]

Antibodies that block cytokine function provide a powerful therapeutic tool especially for the treatment of autoimmune diseases. Cytokines are a group of small hydrophilic glycoproteins that bind their receptors on the cell surface and subsequently activate intracellular signalling cascades, such as the JAK/ STAT pathway. A bulk of evidence has demonstrated that genetic mutations in signalling molecules can cause immunodeficiencies and malignant cell growth. As a result, several drug companies have begun to develop therapeutics that inhibit the function of JAK tyrosine kinases. Currently, two JAK inhibitors, tofacitinib and ruxolitinib, are used in the clinic for treating rheumatoid arthritis and myeloproliferative diseases, respectively. Inhibiting JAK function has been shown to efficiently prevent the uncontrolled growth of cancerous cells and to harness overly active immune cells. In the future, other small molecule compounds are likely to come into clinical use, and intense work is ongoing to develop inhibitors that specifically target the constitutively active mutant JAKs. This MiniReview will summarize the basic features of the JAK/ STAT pathway, its role in human disease and the therapeutic potential of JAK/ STAT inhibitors. [ABSTRACT FROM AUTHOR]

Therapeutic options for psoriasis vulgaris have changed during recent decades with the introduction of biologics. Few nationwide studies are available on psoriasis treatment patterns, and those from Finland predate the use of biologics. The aim of this retrospective, population-based registry study was to identify patients with psoriasis vulgaris and their treatment patterns in the secondary care setting in Finland. The study cohort included 41,456 adults with a diagnosis of psoriasis vulgaris in the public secondary healthcare setting from 2012 through 2018. Data on comorbidities, pharmacotherapy, and phototherapy were collected from nationwide healthcare and drug registries. Patients in the cohort had a wide range of comorbidities, with 14.9% having psoriatic arthritis. Treatment was based largely on topical and conventional systemic medications. Conventional medications were used by 28.9% of patients, and methotrexate was the most common option (20.9%). Biologics were used by 7.3% of patients, mostly as second- and third-line treatment. The use of conventional systemic medications, topical treatments, and phototherapy decreased after the initiation of biologics. This study of psoriasis vulgaris in Finland provides a framework for the development of future care practices. [ABSTRACT FROM AUTHOR]

Furin is one of seven proprotein convertase family members that promote proteolytic maturation of proproteins. It is induced in activated T cells and is reported to process a variety of substrates including the anti-inflammatory cytokine transforming growth factor (TGF)-β1 (refs 2–4), but the non-redundant functions of furin versus other proprotein convertases in T cells are unclear. Here we show that conditional deletion of furin in T cells allowed for normal T-cell development but impaired the function of regulatory and effector T cells, which produced less TGF-β1. Furin-deficient T regulatory (Treg) cells were less protective in a T-cell transfer colitis model and failed to induce Foxp3 in normal T cells. Additionally, furin-deficient effector cells were inherently over-active and were resistant to suppressive activity of wild-type Treg cells. Thus, our results indicate that furin is indispensable in maintaining peripheral tolerance, which is due, at least in part, to its non-redundant, essential function in regulating TGF-β1 production. Targeting furin has emerged as a strategy in malignant and infectious disease. Our results suggest that inhibiting furin might activate immune responses, but may result in a breakdown in peripheral tolerance. [ABSTRACT FROM AUTHOR]

Cytokines play pivotal roles in immunity and inflammation, and targeting cytokines and their receptors is an effective means of treating such disorders. Type I and II cytokine receptors associate with Janus family kinases (JAKs) to effect intracellular signaling. These structurally unique protein kinases play essential and specific roles in immune cell development and function. One JAK, JAK3, has particularly selective functions. Mutations of this kinase underlie severe combined immunodeficiency, indicative of its critical role in the development and function of lymphocytes. Because JAK3 appears not to have functions outside of hematopoietic cells, this kinase has been viewed as an excellent therapeutic target for the development of a new class of immunosuppressive drugs. In fact, several companies are developing JAK3 inhibitors, and Phase II studies are underway. Mutations of Tyk2 cause autosomal recessive hyperIgE syndrome, and in principle, Tyk2 inhibitors might also be useful as immunosuppressive drugs. JAK2 gain-of-function mutations (V617F) underlie a subset of disorders collectively referred to as myeloproliferative diseases and phase 2 trials using JAK inhibitors are underway in this setting. Thus, we are learning a great deal about the feasibility and effectiveness of targeting Janus kinases, and it appears likely that this will be a fruitful strategy in a variety of settings. [ABSTRACT FROM AUTHOR]

The recent elucidation of the multiple molecular mechanisms underlying severe combined immunodeficiency (SCID) is an impressive example of the power of molecular medicine. Analysis of patients and the concomitant generation of animal models mimicking these disorders have quickly provided great insights into the pathophysiology of these potentially devastating illnesses. In this review, we summarize the discoveries that led to the understanding of the role of cytokine receptors and a specific tyrosine kinase, Janus kinase 3 (Jak3), in the pathogenesis of SCID. We discuss how the identification of mutations of Jak3 in autosomal recessive SCID has facilitated the diagnosis of these disorders, offered new insights into the biology of this kinase, permitted new avenues for therapy, and provided the rationale for a generation of a new class of immunosuppressants. [ABSTRACT FROM AUTHOR]

Thousands of organs are transplanted each year and millions of people suffer from autoimmune diseases, which creates a need for an armamentarium of immunosuppressive drugs. Unfortunately, immunosuppressants have unwanted side effects owing, in part, to the fact that they have ubiquitous molecular targets. Cytokines have emerged as important controllers of the immune response, and work during the past decade has identified Janus kinases (JAKs) and signal transducers, and activators of transcription (STATs), as crucial intracellular elements in cytokine signalling. Here, we discuss the potential of the JAK/STAT pathway as a target for new immunosuppressants. In particular, the inhibition of JAK3 seems to be an excellent strategy, because of the selective expression and precise functions of this kinase. [ABSTRACT FROM AUTHOR]

The author focuses on the host cell necrosis in Mycobacterium tuberculosis infected macrophages. Topics discussed include health hazards from tuberculosis, effectiveness of Bacillus Calmette-Guérin (BCG) vaccine for tuberculosis, and longer treatment periods along with high risk of intolerance and side effect in treatment of multidrug-resistance (MDR) infections.

The author discusses a study by N. E. Elliott and colleagues which investigated the gain-of-function mutations in the band 4.1, Ezrin, Radixin, and Moesin (FERM) domain of Janus kinase 3 (JAK3) in T-cell leukemia/lymphoma (ATLL) adult patients. He says that the study demonstrates that some of the patients with ATLL have gain-of-function mutations in JAK3 FERM domain. He notes that the findings emphasize the increasing importance of considering JAK inhibitors in treating hematologic malignancies.

Background. Several studies have reported the association of the proinflammatory cytokine IL-6 with temporal lobe epilepsy (TLE), but the regulation of IL-6 in hippocampal sclerosis (HS) is less studied. Objectives. To analyse IL-6 levels and the IL-6/IL-10 ratio in a larger, well-characterized group of patients with drug-resistant focal epilepsy (DRE), focusing especially on TLE and TLE-associated HS. Methods. IL-6 levels were measured by ELISA in plasma in a cross-sectional cohort of 265 patients comprising TLE with HS (n = 34), TLE without HS (n = 103), extratemporal lobe epilepsy (n = 92), and idiopathic generalized epilepsy (n = 36). Results. The IL-6/IL-10 ratio was higher in TLE with HS than in TLE without HS (3.1 vs. 1.6, p = 0.042), whereas the median levels of IL-6 did not differ among epilepsy types. TLE without HS patients had a higher proportion of increased IL-6 levels than TLE with HS patients (p = 0.021). Additionally, IL-6 levels were higher in patients with right-sided HS than in those with left lateralization (1.71 vs. 0.80 pg/mL, p = 0.04). In TLE with HS patients, IL-6 levels showed a negative correlation with seizure frequency during the last month (r = − 0.342 ; p = 0.047), whereas in TLE without HS patients, the correlation was positive but did not reach significance (r = 0.136 ; p = 0.169). Conclusion. IL-6 levels and the IL-6/IL-10 ratio are differentially regulated among patients with TLE depending on the presence of HS and its lateralization, suggesting that TLE with HS is a distinct category regarding cytokine activation. [ABSTRACT FROM AUTHOR]

Background & Aims: Proprotein convertase subtilisin/kexin type 9 (PCSK9) controls blood cholesterol levels by fostering the LDL receptor (LDLR) degradation in hepatocytes. Additionally, PCSK9 has been suggested to participate in immunoregulation by modulating cytokine production. We studied the immunological role of PCSK9 in Streptococcus pneumoniae bacteraemia in vivo and in a human hepatocyte cell line. Methods: CRISPR/Cas9 mutagenesis was utilized to create pcsk9 knock‐out (KO) zebrafish, which were infected with S pneumoniae to assess the role of PCSK9 for the survival of the fish and in the transcriptomic response of the liver. The direct effects of PCSK9 on the expression of acute‐phase reaction (APR) genes were studied in HepG2 cells. Results: The pcsk9 KO zebrafish lines (pcsk9tpu‐13 and pcsk9tpu‐2,+15) did not show developmental defects or gross phenotypical differences. In the S pneumoniae infected zebrafish, the mortality of pcsk9 KOs was similar to the controls. A liver‐specific gene expression analysis revealed that a pneumococcal challenge upregulated pcsk9, and that the pcsk9 deletion reduced the expression of APR genes, including hepcidin antimicrobial peptide (hamp) and complement component 7b (c7b). Accordingly, silencing PCSK9 in vitro in HepG2 cells using small interfering RNAs (siRNAs) decreased HAMP expression. Conclusions: We demonstrate that PCSK9 is not critical for zebrafish survival in a systemic pneumococcal infection. However, PCSK9 deficiency was associated with the lower expression of APR genes in zebrafish and altered the expression of innate immunity genes in a human hepatocyte cell line. Overall, our data suggest an evolutionarily conserved function for PCSK9 in APR in the liver. [ABSTRACT FROM AUTHOR]

STAT6 is a central mediator of IL-4-induced gene responses. STAT6-mediated transcription is dependent on the C-terminal transcription activation domain (TAD), but the mechanisms by which STAT6 activates transcription are poorly understood. Here, we have identified the staphylococcal nuclease (SN)-like domain and tudor domain containing protein p100 as a STAT6 TAD interacting protein p100 was originally characterized as a transcriptional coactivator for Epstein-Barr virus nuclear antigen 2. STAT6 interacted with p100 in vitro and in vivo. The interaction was mediated by the TAD domain of STAT6 and the SN-like domain of p100. p100 did not affect the immediate activation events of STAT6, but enhanced STAT6-mediated transcriptional activation and the IL-4-induced Igε gene transcription in human B-cell tine. Finally, p100 associated with the large subunit of RNA potymerase II and was mediating interaction between STAT6 and RNA polymerase II. These findings identify p100 as a novel coactivator for STAT6 and suggest that p100 functions as a bridging factor between STAT6 and the basal transcription machinery. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the effects of deep brain stimulation of the anterior nucleus of the thalamus (ANT-DBS) on systemic inflammatory responses in patients with drug-resistant epilepsy (DRE). Twenty-two Finnish patients with ANT-DBS implantation were enrolled in this pilot study. Changes in plasma interleukin-6 (IL-6) and interleukin-10 (IL-10) levels were examined using generalized estimating equation models at seven time points (before DBS surgery and 1, 2, 3, 6, 9 and 12 months after implantation). In the whole group, the IL-6/IL-10 ratio decreased significantly over time following ANT-DBS, while the decrease in IL-6 levels and increase in IL-10 levels were not significant. In the responder and nonresponder groups, IL-6 levels remained unchanged during the follow-up. Responders had significantly lower pre-DBS IL-10 levels before the ANT-DBS treatment than nonresponders, but the levels significantly increased over time after the treatment. In addition, responders had a higher pre-DBS IL-6/IL-10 ratio than nonresponders, and the ratio decreased for both groups after treatment, but the decrease did not reach the level of statistical significance. The rate of decrease in the ratio per month tended to be higher in responders than in nonresponders. These results may highlight the anti-inflammatory properties of ANT-DBS treatment associated with its therapeutic effectiveness in patients with DRE. Additional studies are essential to evaluate the potential of the proinflammatory cytokine IL-6, the anti-inflammatory cytokine IL-10, and their ratio as biomarkers to evaluate the therapeutic response to DBS treatment, which could facilitate treatment optimization. [ABSTRACT FROM AUTHOR]

Background: Virus-like particle (VLP) vaccines have recently emerged as a safe and effective alternative to conventional vaccine technologies. The strong immunogenic effects of VLPs can be harnessed for making vaccines against any pathogen by decorating VLPs with antigens from the pathogen. Producing the antigenic pathogen fragments and the VLP platform separately makes vaccine development rapid and convenient. Here we decorated the norovirus-like particle with two conserved influenza antigens and tested for the immunogenicity of the vaccine candidates in BALB/c mice. Results: SpyTagged noro-VLP was expressed with high efficiency in insect cells and purified using industrially scalable methods. Like the native noro-VLP, SpyTagged noro-VLP is stable for months when refrigerated in a physiological buffer. The conserved influenza antigens were produced separately as SpyCatcher fusions in E. coli before covalent conjugation on the surface of noro-VLP. The noro-VLP had a high adjuvant effect, inducing high titers of antibody production against the antigens presented on its surface. Conclusions: The modular noro-VLP vaccine platform presented here offers a rapid, convenient and safe method to present various soluble protein antigens to the immune system for vaccination and antibody production purposes. [ABSTRACT FROM AUTHOR]

Abstract: Interleukin-7 is a homeostatic cytokine that contributes to the maintenance of the T cell pool. It also has proinflammatory effects and is involved in the pathogenesis of autoimmune diseases. Due to its homeostatic effects, IL-7 has been proposed as a potential rejuvenation factor for the elderly immune system. We analyzed the correlation of plasma IL-7 concentrations and the proportions of different T cell populations in nonagenarians (n=163) participating in the Vitality 90+ study. Young individuals (n=35, aged 19–30years) were used as controls. The numbers of CD3+, CD14+, CD4+ and CD8+ cells and the expression of the CD28 costimulatory molecule on CD4+ and CD8+ lymphocyte subsets were analyzed using flow cytometry. The plasma IL-7 levels were significantly higher in the nonagenarians compared to the controls (7.86 vs. 5.74pg/ml, p=0.004). In the nonagenarians, plasma IL-7 levels correlated inversely with the proportion of CD3+ T cells and directly with the proportion of CD14+ monocytes and plasma C-reactive protein. No correlation was observed between plasma IL-7 levels and the proportions of CD4+CD28− or CD8+CD28− subsets. These results suggest that the IL-7 levels in nonagenarians do not have an inhibitory effect on the development of immunosenescence; rather they are associated with increased inflammation. [Copyright &y& Elsevier]

Ischemic retinopathy is a vision-threatening disease associated with chronic retinal inflammation and hypoxia leading to abnormal angiogenesis. Furin, a member of the proprotein convertase family of proteins, has been implicated in the regulation of angiogenesis due to its essential role in the activation of several angiogenic growth factors, including vascular endothelial growth factor-C (VEGF-C), VEGF-D and transforming growth factor - β (TGF- β). In the present study, we evaluated expression of furin in the retina and its role in retinal angiogenesis. As both inflammation and hypoxia contribute to angiogenesis, the role of furin was evaluated using myeloid-cell specific furin knockout (KO) mice (designated LysMCre-fur (fl/fl) ) both in developmental retinal angiogenesis as well as in hypoxia-driven angiogenesis using the oxygen-induced retinopathy (OIR) model. In the retina, furin expression was detected in endothelial cells, macrophages and, to some extent, in neurons. The rate of angiogenesis was not different in LysMCre-fur (fl/fl) mice when compared to their wild-type littermates during development. In the OIR model, the revascularization of retina was significantly delayed in LysMCre-fur (fl/fl) mice compared to their wild-type littermates, while there was no compensatory increase in the preretinal neovascularization in LysMCre-fur (fl/fl) mice. These results demonstrate that furin expression in myeloid cells plays a significant role in hypoxia-induced angiogenesis in retina. [ABSTRACT FROM AUTHOR]

Invading pathogens provoke robust innate immune responses in Dipteran insects, such as Drosophila melanogaster. In a systemic bacterial infection, a humoral response is induced in the fat body. Gram-positive bacteria trigger the Toll signaling pathway, whereas gram-negative bacterial infections are signaled via the immune deficiency (IMD) pathway. We show here that the RNA interference-mediated silencing of Furin1--a member of the proprotein convertase enzyme family--specifically in the fat body, results in a reduction in the expression of antimicrobial peptides. This, in turn, compromises the survival of adult fruit flies in systemic infections that are caused by both gram-positive and -negative bacteria. Furin1 plays a nonredundant role in the regulation of immune responses, as silencing of Furin2, the other member of the enzyme family, had no effect on survival or the expression of antimicrobial peptides upon a systemic infection. Furin1 does not directly affect the Toll or IMD signaling pathways, but the reduced expression of Furin1 up-regulates stress response factors in the fat body. We also demonstrate that Furin1 is a negative regulator of the Janus kinase/signal transducer and activator of transcription signaling pathway, which is implicated in stress responses in the fly. In summary, our data identify Furin1 as a novel regulator of humoral immunity and cellular stress responses in Drosophila. [ABSTRACT FROM AUTHOR]

Introduction: Hip arthroplasty is the standard treatment of a painful hip destruction. The use of modern metal-on-metal (MOM) bearing surfaces gained popularity in total hip arthroplasties during the last decade. Recently, worrisome failures due to adverse reaction to metal debris (ARMD), including pseudotumor response, have been widely reported. However, the pathogenesis of this reaction remains poorly understood. The aim of the present study was to investigate the ARMD response by flow cytometry approach. Methods: Sixteen patients with a failed Articular Surface Replacement (ASR) hip prosthesis were included in the study. Samples of pseudotumor tissues collected during revision surgery were degraded by enzyme digestion and cells were typed by flow cytometry. Whole blood chromium and cobalt concentrations were analyzed with mass spectrometry before revision surgery. Results: Flow cytometry analysis showed that the peri-implant pseudotumor tissue expressed two principal phenotypes, namely macrophage-dominated and T-lymphocyte-dominated response; the average portions being 54% (macrophages) and 25% (T-lymphocytes) in macrophage-dominated inflammation and 20% (macrophages) and 54% (T-lymphocytes) in T-lymphocyte-dominated response. The percentages of B-lymphocytes and granulocytes were lower in both phenotypes. Interestingly, the levels of blood chromium and cobalt were significantly higher in patients with macrophage-dominated response. Conclusions: The results suggest that the adverse tissue reactions induced by MOM wear particles contain heterogeneous pathogeneses and that the metal levels are an important factor in the determination of the inflammatory phenotype. The present results support the hypothesis that higher metal levels cause cytotoxicity and tissue injury and macrophages are recruited to clear the necrotic debris. On the other hand, the adverse response developed in association with lower metal levels is T-lymphocyte-dominated and is likely to reflect hypersensitivity reaction. [ABSTRACT FROM AUTHOR]

Proprotein convertase subtilisin/kexin (PCSK) enzymes cleave and convert their immature substrates into biologically active forms. Polymorphisms in the PCSK genes have been reported to associate with human diseases and phenotypes, including hypercholesterolemia and blood pressure (BP), and targeting PCSKs is considered a promising future form of drug therapy. PCSK processing is readily induced upon upregulation of the enzyme, but the genetic factors contributing to PCSK expression have not been thoroughly characterized. To gain a comprehensive understanding of the genetic regulation of PCSK expression, we performed, for the first time, a genome-wide expression quantitative trait loci (eQTL) analysis using mRNA expression in >1400 human peripheral blood samples from the Cardiovascular Risk in Young Finns Study and ca. ten million single-nucleotide polymorphisms (SNPs). The expression data showed clear expression for FURIN, PCSK5, PCSK7 and MBTPS1 (membrane-bound transcription factor peptidase, site 1) mRNAs in virtually all tested samples. A discovery analysis demonstrated a genome-wide significant ( p < 5 × 10) association with the selected PCSK probes for 1024 variants, which were located at ten independent loci. Of these loci, 5/10 could be confirmed to regulate PCSK expression in two additional and independent sample sets. Finally, a phenotypic analysis demonstrated that a novel cis-eQTL SNP rs4702 for FURIN is strongly associated with both diastolic ( p = 0.012) and systolic ( p = 0.035) BP levels, as well as peripheral vascular resistance ( p = 0.003). These findings indicate that the expression of the PCSK enzymes is regulated by genetic factors, which have biological roles in health and disease. [ABSTRACT FROM AUTHOR]

The proprotein convertase subtilisin/kexin enzymes proteolytically convert immature proproteins into bioactive molecules, and thereby they serve as key regulators of cellular homeostasis. The archetype proprotein convertase subtilisin/kexin, FURIN, is a direct target gene of the IL-12/STAT4 pathway and it is upregulated in Th1 cells. We have previously demonstrated that FURIN expression in T cells critically regulates the maintenance of peripheral immune tolerance and the functional maturation of pro-TGF-ß1 in vivo, but FURIN's role in cell-mediated immunity and Th polarization has remained elusive. In this article, we show that T cell-expressed FURIN is essential for host resistance against a prototypic Th1 pathogen, Toxoplasma gondii, and for the generation of pathogen-specific Th1 lymphocytes, including Th1-IL-10 cells. FURIN-deficient Th cells instead show elevated expression of IL-4R subunit a on cell surface, sensitized IL-4/STAT6 signaling, and a propensity to polarize toward the Th2 phenotype. By exploring FURIN-interacting proteins in Jurkat T cells with Strep-Tag purification and mass spectrometry, we further identify an association with a cytoskeleton modifying Ras-related C3 botulinum toxin substrate/dedicator of cytokinesis 2 protein complex and unravel that FURIN promotes F-actin polymerization, which has previously been shown to downregulate IL-4R subunit a cell surface expression and promote Th1 responses. In conclusion, our results demonstrate that in addition to peripheral immune tolerance, T cell-expressed FURIN is also a central regulator of cell-mediated immunity and Th1/2 cell balance. [ABSTRACT FROM AUTHOR]

Tuberculosis is still a major health problem worldwide. Currently it is not known what kind of immune responses lead to successful control and clearance of Mycobacterium tuberculosis. This gap in knowledge is reflected by the inability to develop sufficient diagnostic and therapeutic tools to fight tuberculosis. We have used the Mycobacterium marinum infection model in the adult zebrafish and taken advantage of heterogeneity of zebrafish population to dissect the characteristics of adaptive immune responses, some of which are associated with well-controlled latency or bacterial clearance while others with progressive infection. Differences in T cell responses between subpopulations were measured at the transcriptional level. It was discovered that a high total T cell level was usually associated with lower bacterial loads alongside with a T helper 2 (Th2)-type gene expression signature. At late time points, spontaneous reactivation with apparent symptoms was characterized by a low Th2/Th1 marker ratio and a substantial induction of foxp3 reflecting the level of regulatory T cells. Characteristic gata3/tbx21 has potential as a biomarker for the status of mycobacterial disease. [ABSTRACT FROM AUTHOR]

Proprotein convertase subtilisin/kexin (PCSK) enzymes convert proproteins into bioactive end products. Although other PCSK enzymes are known to be essential for biological processes ranging from cholesterol metabolism to host defense, the in vivo importance of the evolutionarily ancient PCSK7 has remained enigmatic. Here, we quantified the expressions of all pcsk genes during the 1st week of fish development and in several tissues. pcsk7 expression was ubiquitous and evident already during the early development. To compare mammalian and zebrafish PCSK7, we prepared homology models, which demonstrated remarkable structural conservation. When the PCSK7 function in developing larvae was inhibited, we found that PCSK7-deficient fish have defects in various organs, including the brain, eye, and otic vesicle, and these result in mortality within 7 days postfertilization. A genome-wide analysis of PCSK7-dependent gene expression showed that, in addition to developmental processes, several immune system-related pathways are also regulated by PCSK7. Specifically, the PCSK7 contributed to the mRNA expression and proteolytic cleavage of the cytokine TGFβ1a. Consequently, tgfβ1a morphant fish displayed phenotypical similarities with pcsk7 morphants, underscoring the importance of this cytokine in the zebrafish development. Targeting PCSK activity has emerged as a strategy for treating human diseases. Our results suggest that inhibiting PCSK7 might interfere with normal vertebrate development. [ABSTRACT FROM AUTHOR]

The ability to positively affect host health through the modulation of the immune response is a feature of increasing importance in measuring the probiotic potential of a bacterial strain. However, the identities of the bacterial cell components involved in cross talk with immune cells remain elusive. In this study, we characterized the dairy strain Lactobacillus helveticus MIMLh5 and its surface-layer protein (SlpA) using in vitro and ex vivo analyses. We found that MIMLh5 and S1pA exert anti-inflamma- tory effects by reducing the activation of NF-κB on the intestinal epithelial Caco-2 cell line. On the contrary, MIMLh5 and S1pA act as stimulators of the innate immune system by triggering the expression of proinflammatory factors tumor necrosis factor alpha and COX-2 in the human macrophage cell line U937 via recognition through Toll-like receptor 2. In the same experiments, SlpA protein did not affect the expression of the anti-inflammatory cytokine interleukin-10. A similar response was observed following stimulation of macrophages isolated from mouse bone marrow or the peritoneal cavity. These results suggest that S1pA plays a major role in mediating bacterial immune-stimulating activity, which could help to induce the host's defenses against and responses toward infections. This study supports the concept that the viability of bacterial cells is not always essential to exert immunomodulatory effects, thus permitting the development of safer therapies for the treatment of specific diseases according to a paraprobiotic intervention. [ABSTRACT FROM AUTHOR]

The mechanisms leading to latency and reactivation of human tuberculosis are still unclear, mainly due to the lack of standardized animal models for latent mycobacterial infection. In this longitudinal study of the progression of a mycobacterial disease in adult zebrafish, we show that an experimental intraperitoneal infection with a low dose (~35 bacteria) of Mycobacterium marinum, results in the development of a latent disease in most individuals. The infection is characterized by limited mortality (25%), stable bacterial loads 4 weeks following infection and constant numbers of highly organized granulomas in few target organs. The majority of bacteria are dormant during a latent mycobacterial infection in zebrafish, and can be activated by resuscitation promoting factor ex vivo. In 5-10% of tuberculosis cases in humans, the disease is reactivated usually as a consequence of immune suppression. In our model, we are able to show that reactivation can be efficiently induced in infected zebrafish by γ-irradiation that transiently depletes granulo/monocyte and lymphocyte pools, as determined by flow cytometry. This immunosuppression causes reactivation of the dormant mycobacterial population and a rapid outgrowth of bacteria, leading to 88% mortality in four weeks. In this study, the adult zebrafish presents itself as a unique non-mammalian vertebrate model for studying the development of latency, regulation of mycobacterial dormancy, as well as reactivation of latent or subclinical tuberculosis. The possibilities for screening for host and pathogen factors affecting the disease progression, and identifying novel therapeutic agents and vaccine targets make this established model especially attractive. [ABSTRACT FROM AUTHOR]

The use of proper bacterial strains as probiotics for the pharyngeal mucosa is a potential prophylactic strategy for upper respiratory tract infections. In this context, we characterized in vitro the functional and immunomodulatory properties of the strains Lactobacillus helveticus MIMLh5 and Streptococcus salivarius ST3 that were selected during previous investigations as promising pharyngeal probiotics. In this study, we demonstrated in vitro that strains MIMLh5 and ST3, alone and in combination, can efficiently adhere to pharyngeal epithelial cells, antagonize Streptococcus pyogenes, and modulate host innate immunity by inducing potentially protective effects. In particular, we found that the strains MIMLh5 and ST3 activate U937 human macrophages by significantly inducing the expression of the proinflammatory cytokine tumor necrosis factor alpha (TNF-a). Nonetheless, the induction of the anti-inflammatory interleukin-10 (IL-10) by MIMLh5 or ST3 was never lower than that of TNF-a, suggesting that these bacteria can potentially exert a regulatory rather than a proinflammatory effect. We also found that the strains MIMLh5 and ST3 induce cyclooxygenase 2 (COX-2) expression and demonstrated that toll-like receptor 2 (TLR-2) participates in the recognition of the strains MIMLh5 and ST3 by U937 cells. Finally, we observed that these microorganisms grow efficiently when cocultured in milk, suggesting that the preparation of a milk-based fermented product containing both MIMLh5 and ST3 can be a practical solution for the administration of these bacteria. In conclusion, we propose the combined use of L. helveticus MIMLh5 and S. salivarius ST3 for the preparation of novel products that display probiotic properties for the pharyngeal mucosa. [ABSTRACT FROM AUTHOR]

Abstract: Background: Proprotein convertase subtilisin/kexin (PCSK) enzymes cleave proproteins into mature end products. Previously, MBTPS1 and PCSK9 have been shown to regulate cholesterol metabolism and LDL receptor recycling, whereas FURIN and PCSK5 have been suggested to inactivate lipases and regulate inflammation in atherosclerosis. Here, we systematically analyzed the expression of PCSKs and their targets in advanced atherosclerotic plaques. Methods and results: Microarray and quantitative real-time PCR experiments showed that FURIN (42.86 median fold, p =2.1e−8), but no other PCSK, is universally overexpressed in the plaques of different vascular regions. The mRNA expression screen of PCSK target proteins in plaques identified many known factors, but it also identified the significant upregulation of the previously overlooked furin-processed B cell activating cytokines APRIL (TNFSF13, 2.52 median fold, p =3.0e−5) and BAFF (TNFSF13B, 2.97 median fold, p =7.6e−6). The dysregulation of FURIN did not associate with its htSNPs or the previously reported regulatory SNP (−229, rs4932178) in the promoter. Immunohistochemistry experiments showed the upregulation of FURIN in the plaque lymphocytes and macrophages where it was co-expressed with BAFF/TNFSF13B and APRIL/TNFSF13. Conclusions: Our data unequivocally show that FURIN is the primary PCSK that is dysregulated in the immune cells of advanced human atherosclerotic plaques, which implies a role for this enzyme in plaque pathology. Therefore, drugs that inhibit FURIN in arteries may modulate the course of this disease. [Copyright &y& Elsevier]

Intestinal microfold cells (M cells) are a dynamic lineage of epithelial cells that initiate mucosal immunity in the intestine. They are responsible for the uptake and transcytosis of microorganisms, pathogens, and other antigens in the gastrointestinal tract. A mature M cell expresses a receptor Gp2 which binds to pathogens and aids in the uptake. Due to the rarity of these cells in the intestine, their development and differentiation remain yet to be fully understood. We recently demonstrated that polycomb repressive complex 2 (PRC2) is an epigenetic regulator of M cell development, and 12 novel transcription factors including Atoh8 were revealed to be regulated by the PRC2. Here, we show that Atoh8 acts as a regulator of M cell differentiation; the absence of Atoh8 led to a significant increase in the number of Gp2+ mature M cells and other M cell-associated markers such as Spi-B and Sox8. In vitro organoid analysis of RankL treated organoid showed an increase of mature marker GP2 expression and other M cell-associated markers. Atoh8 null mice showed an increase in transcytosis capacity of luminal antigens. An increase in M cell population has been previously reported to be detrimental to mucosal immunity because some pathogens like orally acquired prions have been able to exploit the transcytosis capacity of M cells to infect the host; mice with an increased population of M cells are also susceptible to Salmonella infections. Our study here demonstrates that PRC2 regulated Atoh8 is one of the factors that regulate the population density of intestinal M cell in the Peyer's patch. [ABSTRACT FROM AUTHOR]

Tuberculosis is a multifactorial bacterial disease, which can be modeled in the zebrafish (Danio rerio). Abdominal cavity infection with Mycobacterium marinum, a close relative of Mycobacterium tuberculosis, leads to a granulomatous disease in adult zebrafish, which replicates the different phases of human tuberculosis, including primary infection, latency and spontaneous reactivation. Here, we have carried out a transcriptional analysis of zebrafish challenged with low-dose of M. marinum, and identified intelectin 3 (itln3) among the highly up-regulated genes. In order to clarify the in vivo significance of Itln3 in immunity, we created nonsense itln3 mutant zebrafish by CRISPR/Cas9 mutagenesis and analyzed the outcome of M. marinum infection in both zebrafish embryos and adult fish. The lack of functional itln3 did not affect survival or the mycobacterial burden in the zebrafish. Furthermore, embryonic survival was not affected when another mycobacterial challenge responsive intelectin, itln1, was silenced using morpholinos either in the WT or itln3 mutant fish. In addition, M. marinum infection in dexamethasone-treated adult zebrafish, which have lowered lymphocyte counts, resulted in similar bacterial burden in both WT fish and homozygous itln3 mutants. Collectively, although itln3 expression is induced upon M. marinum infection in zebrafish, it is dispensable for protective mycobacterial immune response. [ABSTRACT FROM AUTHOR]

Proprotein convertases (PCSK) have a critical role in the body homeostasis as enzymes responsible for processing precursor proteins into their mature forms. FURIN, the first characterized member of the mammalian PCSK family, is overexpressed in multiple malignancies and the inhibition of its activity has been considered potential cancer treatment. FURIN has also an important function in the adaptive immunity, since its deficiency in T cells causes an impaired peripheral immune tolerance and accelerates immune responses. We addressed whether deleting FURIN from the immune cells would strengthen anticancer responses by subjecting mouse strains lacking FURIN from either T cells or macrophages and granulocytes to the DMBA/TPA two-stage skin carcinogenesis protocol. Unexpectedly, deficiency of FURIN in T cells resulted in enhanced and accelerated development of tumors, whereas FURIN deletion in macrophages and granulocytes had no effect. The epidermises of T-cell-specific FURIN deficient mice were significantly thicker with more proliferating Ki67+ cells. In contrast, there were no differences in the numbers of the T cells. The flow cytometric analyses of T-cell populations in skin draining lymph nodes showed that FURIN T-cell KO mice have an inherent upregulation of early activation marker CD69 as well as more CD4+CD25+Foxp3+positive T regulatory cells. In the early phase of tumor promotion, T cells from the T-cell-specific FURIN knockout animals produced more interferon gamma, whereas at later stage the production of Th2- and Th17-type cytokines was more prominent than in wild-type controls. In conclusion, while PCSK inhibitors are promising therapeutics in cancer treatment, our results show that inhibiting FURIN specifically in T cells may promote squamous skin cancer development. [ABSTRACT FROM AUTHOR]

The avidin protein family members are well known for their high affinity towards D-biotin and high structural stability. These properties make avidins valuable tools for a wide range of biotechnology applications. We have identified a new member of the avidin family in the zebrafish (Danio rerio) genome, hereafter called zebavidin. The protein is highly expressed in the gonads of both male and female zebrafish and in the gills of male fish, but our data suggest that zebavidin is not crucial for the developing embryo. Biophysical and structural characterisation of zebavidin revealed distinct properties not found in any previously characterised avidins. Gel filtration chromatography and native mass spectrometry suggest that the protein forms dimers in the absence of biotin at low ionic strength, but assembles into tetramers upon binding biotin. Ligand binding was analysed using radioactive and fluorescently labelled biotin and isothermal titration calorimetry. Moreover, the crystal structure of zebavidin in complex with biotin was solved at 2.4 Å resolution and unveiled unique ligand binding and subunit interface architectures; the atomic-level details support our physicochemical observations. [ABSTRACT FROM AUTHOR]