Viperin, an IFN-Stimulated Protein, Delays Rotavirus Release by Inhibiting Non-Structural Protein 4 (NSP4)-Induced Intrinsic Apoptosis.

Viral infections lead to expeditious activation of the host's innate immune responses, most importantly the interferon (IFN) response, which manifests a network of interferon-stimulated genes (ISGs) that constrain escalating virus replication by fashioning an ill-disposed environment. Interestingly, most viruses, including rotavirus, have evolved numerous strategies to evade or subvert host immune responses to establish successful infection. Several studies have documented the induction of ISGs during rotavirus infection. In this study, we evaluated the induction and antiviral potential of viperin, an ISG, during rotavirus infection. We observed that rotavirus infection, in a stain independent manner, resulted in progressive upregulation of viperin at increasing time points post-infection. Knockdown of viperin had no significant consequence on the production of total infectious virus particles. Interestingly, substantial escalation in progeny virus release was observed upon viperin knockdown, suggesting the antagonistic role of viperin in rotavirus release. Subsequent studies unveiled that RV-NSP4 triggered relocalization of viperin from the ER, the normal residence of viperin, to mitochondria during infection. Furthermore, mitochondrial translocation of NSP4 was found to be impeded by viperin, leading to abridged cytosolic release of Cyt c and subsequent inhibition of intrinsic apoptosis. Additionally, co-immunoprecipitation studies revealed that viperin associated with NSP4 through regions including both its radical SAM domain and its C-terminal domain. Collectively, the present study demonstrated the role of viperin in restricting rotavirus egress from infected host cells by modulating NSP4 mediated apoptosis, highlighting a novel mechanism behind viperin's antiviral action in addition to the intricacy of viperin-virus interaction.

Retinol Saturase Knock-Out Mice are Characterized by Impaired Clearance of Apoptotic Cells and Develop Mild Autoimmunity.

Apoptosis and the proper clearance of apoptotic cells play a central role in maintaining tissue homeostasis. Previous work in our laboratory has shown that when a high number of cells enters apoptosis in a tissue, the macrophages that engulf them produce retinoids to enhance their own phagocytic capacity by upregulating several phagocytic genes. Our data indicated that these retinoids might be dihydroretinoids, which are products of the retinol saturase (RetSat) pathway. In the present study, the efferocytosis of RetSat-null mice was investigated. We show that among the retinoid-sensitive phagocytic genes, only transglutaminase 2 responded in macrophages and in differentiating monocytes to dihydroretinol. Administration of dihydroretinol did not affect the expression of the tested genes differently between differentiating wild type and RetSat-null monocytes, despite the fact that the expression of RetSat was induced. However, in the absence of RetSat, the expression of numerous differentiation-related genes was altered. Among these, impaired production of MFG-E8, a protein that bridges apoptotic cells to the αvß3/ß5 integrin receptors of macrophages, resulted in impaired efferocytosis, very likely causing the development of mild autoimmunity in aged female mice. Our data indicate that RetSat affects monocyte/macrophage differentiation independently of its capability to produce dihydroretinol at this stage.

Deletion of Biliverdin Reductase A in Myeloid Cells Promotes Chemokine Expression and Chemotaxis in Part via a Complement C5a--C5aR1 Pathway.

Biliverdin reductase (BVR)-A is a pleotropic enzyme converting biliverdin to bilirubin and a signaling molecule that has cytoprotective and immunomodulatory effects. We recently showed that biliverdin inhibits the expression of complement activation fragment 5a receptor one (C5aR1) in RAW 264.7 macrophages. In this study, we investigated the role of BVR-A in determining macrophage inflammatory phenotype and function via regulation of C5aR1. We assessed expression of C5aR1, M1-like macrophage markers, including chemokines (RANTES, IP-10), as well as chemotaxis in response to LPS and C5a in bone marrow-derived macrophages from BVR fl/fl and LysM-Cre:BVR fl / fl mice (conditional deletion of BVR-A in myeloid cells). In response to LPS, macrophages isolated from LysM-Cre:BVR fl/fl showed significantly elevated levels of C5aR1 as well as chemokines (RANTES, IP10) but not proinflammatory markers, such as iNOS and TNF. An increase in C5aR1 expression was also observed in peritoneal macrophages and several tissues from LysM-Cre:BVR fl/fl mice in a model of endotoxemia. In addition, knockdown of BVR-A resulted in enhanced macrophage chemotaxis toward C5a. Part of the effects of BVR-A deletion on chemotaxis and RANTES expression were blocked in the presence of a C5aR1 neutralizing Ab, confirming the role of C5a-C5aR1 signaling in mediating the effects of BVR. In summary, BVR-A plays an important role in regulating macrophage chemotaxis in response to C5a via modulation of C5aR1 expression. In addition, macrophages lacking BVR-A are characterized by the expression of M1 polarization-associated chemokines, the levels of which depend in part on C5aR1 signaling.

Use of human Dihydroorotate Dehydrogenase (hDHODH) Inhibitors in Autoimmune Diseases and New Perspectives in Cancer Therapy.

BACKGROUND: Human dihydroorotate dehydrogenase (hDHODH, EC 1.3.5.2), a flavindependent mitochondrial enzyme involved in de novo pyrimidine biosynthesis, is a validated therapeutic target for the treatment of autoimmune diseases, such as rheumatoid arthritis and multiple sclerosis. However, human DHODH inhibitors have also been investigated as treatment for cancer, parasite infections (i.e. malaria) and viruses as well as in the agrochemicals industry. OBJECTIVE: An overview of current knowledge of hDHODH inhibitors and their potential uses in diseases where hDHODH is involved. METHOD: This review focuses on recent advances in the development and application of hDHODH inhibitors, specifically covering the patent field, starting from a brief description of enzyme topography and of the strategies usually followed in designing its selective inhibitors. RESULTS: The most important and well-described novelty is the fact that the discovery, in the autumn of 2016, that hDHODH inhibitors are able to induce in vivo myeloid differentiation has led to the possibility of developing novel hDHODH based treatments for Acute Myelogenous Leukemia (AML). CONCLUSION: The review will describe a variety of specific inhibitor classes and conclude on recent and future therapeutic perspectives for this target.

Quantitative proteome analysis of bovine mammary gland reveals protein dynamic changes involved in peak and late lactation stages.

Mammary gland is an important organ for milk synthesis and secretion. It undergoes dramatic physiological changes to adapt the shift from peak to late lactation stage. Protein plays a final very vital role in many life functions, and the protein changes during different lactation stages potentially reflect the biology of lactation and the functions of mammary gland in cows. In current study, we adopted tandem mass tags label-based quantitative analysis technique and to investigate proteome changes occurring in bovine mammary gland from peak to late lactation stages. A total of 3753 proteins from mammary tissues taken at two lactation points from four individual cows by biopsy were quantified, out of which 179 proteins were expressed differentially between two stages. We observed five new DEPs (AACS, DHCR7, GSTM3, SFRP1 and SFRP4) and nine functional well-studies known proteins (PLIN2, LPIN1, PLIN3, GSN, CD74, MMP2, SOD1, SOD3 and GPX3) related to milk performance and mammary morphology. Bioinformatics analyses of the DEPs showed a majority of the up-regulated proteins during late lactation stage were related to apoptosis and immune process, while the downregulated proteins were mainly involved in localization, lipid metabolic and transport process. This suggests that the mammary gland can adapt to different molecular functions according to the biological need of the animal. From the integrated analysis of the differentially expressed proteins with known quantitative trait loci and genome-wide association study data, we identified 95 proteins may potentially affect milking performance. We expect findings in this study could be a valuable resource for future studies investigating the bovine proteome and functional studies.

Mining Novel Allergens from Coconut Pollen Employing Manual De Novo Sequencing and Homology-Driven Proteomics.

Coconut pollen, one of the major palm pollen grains is an important constituent among vectors of inhalant allergens in India and a major sensitizer for respiratory allergy in susceptible patients. To gain insight into its allergenic components, pollen proteins were analyzed by two-dimensional electrophoresis, immunoblotted with coconut pollen sensitive patient sera, followed by mass spectrometry of IgE reactive proteins. Coconut being largely unsequenced, a proteomic workflow has been devised that combines the conventional database-dependent analysis of tandem mass spectral data and manual de novo sequencing followed by a homology-based search for identifying the allergenic proteins. N-terminal acetylation helped to distinguish "b" ions from others, facilitating reliable sequencing. This led to the identification of 12 allergenic proteins. Cluster analysis with individual patient sera recognized vicilin-like protein as a major allergen, which was purified to assess its in vitro allergenicity and then partially sequenced. Other IgE-sensitive spots showed significant homology with well-known allergenic proteins such as 11S globulin, enolase, and isoflavone reductase along with a few which are reported as novel allergens. The allergens identified can be used as potential candidates to develop hypoallergenic vaccines, to design specific immunotherapy trials, and to enrich the repertoire of existing IgE reactive proteins.

Biliverdin Reductase A (BVRA) Mediates Macrophage Expression of Interleukin-10 in Injured Kidney.

Biliverdin reductase A is an enzyme, with serine/threonine/tyrosine kinase activation, converting biliverdin (BV) to bilirubin (BR) in heme degradation pathway. It has been reported to have anti-inflammatory and antioxidant effect in monocytes and human glioblastoma. However, the function of BVRA in polarized macrophage was unknown. This study aimed to investigate the effect of BVRA on macrophage activation and polarization in injured renal microenvironment. Classically activated macrophages (M1macrophages) and alternative activation of macrophages (M2 macrophages) polarization of murine bone marrow derived macrophage was induced by GM-CSF and M-CSF. M1 polarization was associated with a significant down-regulation of BVRA and Interleukin-10 (IL-10), and increased secretion of TNF-α. We also found IL-10 expression was increased in BVRA over-expressed macrophages, while it decreased in BVRA knockdown macrophages. In contrast, BVRA over-expressed or knockdown macrophages had no effect on TNF-α expression level, indicating BVRA mediated IL-10 expression in macrophages. Furthermore, we observed in macrophages infected with recombinant adenoviruses BVRA gene, which BVRA over-expressed enhanced both INOS and ARG-1 mRNA expression, resulting in a specific macrophage phenotype. Through in vivo study, we found BVRA positive macrophages largely existed in mice renal ischemia perfusion injury. With the treatment of the regular cytokines GM-CSF, M-CSF or LPS, excreted in the injured renal microenvironment, IL-10 secretion was significantly increased in BVRA over-expressed macrophages. In conclusion, the BVRA positive macrophage is a source of anti-inflammatory cytokine IL-10 in injured kidney, which may provide a potential target for treatment of kidney disease.

Serum DHCR24 Auto-antibody as a new Biomarker for Progression of Hepatitis C.

BACKGROUND: New biomarkers are needed to identify the stage of hepatitis C virus (HCV)-infected diseases in order to reduce the mortality rates. Herein, we investigated whether serum 3ß-hydroxysterol Δ24-reductase antibody (DHCR24 Ab) may serve as a prognostic marker for hepatitis C infection progression to hepatocellular carcinoma (HCC). METHODS: Serum DHCR24 Abs from 395 HCV-positive patients, including 133 chronic hepatitis (CHC), 85 liver cirrhosis (LCC), and 177 HCC (HCC-C) patients; 232 hepatitis B virus (HBV)-positive patients, including 103 chronic hepatitis (CHB), 56 liver cirrhosis (LCB), and 73 HCC (HCC-B) patients; and 24 healthy controls, were measured using enzyme-linked immunosorbent assay. RESULTS: The serum DHCR24 Ab levels were significantly higher in patients with CHC than in healthy controls, in LCC than in CHC, and in LCC than in HCC-C (P < 0.0001 for all). The concentration of serum DHCR24 Ab in HCC-B patients showed no significant difference compared to CHB and LCB patients (P = 0.1247). The DHCR24 Ab levels were significantly higher in early HCC-C than CHC or LCC patients and in late HCC-C compared to early HCC-C patients. The sensitivity of the DHCR24 Ab for HCC-C detection (70.6%) was higher than that of alpha-fetoprotein (AFP; 54.8%) and protein induced by vitamin K absence or antagonist-II (PIVKA-II; 42 · 5%). Moreover, DHCR24 was up-regulated in HCV-positive, but not HBV-positive tissues or HBV-negative, HCV-negative HCC specimens. CONCLUSIONS: DHCR24 auto-antibody represents a potential noninvasive biomarker for HCV-related liver disease and may facilitate the diagnosis of PIVKA-II and AFP-negative HCC.

3ß-hydroxysterol δ24-reductase on the surface of hepatitis C virus-related hepatocellular carcinoma cells can be a target for molecular targeting therapy.

In our previous study, we demonstrated that 3ß-hydroxysterol Δ24-reductase (DHCR24) was overexpressed in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC), and that its expression was induced by HCV. Using a monoclonal antibody against DHCR24 (2-152a MAb), we found that DHCR24 was specifically expressed on the surface of HCC cell lines. Based on these findings, we aimed to establish a novel targeting strategy using 2-152a MAb to treat HCV-related HCC. In the present study, we examined the antitumor activity of 2-152a MAb. In the presence of complement, HCC-derived HuH-7 cells were killed by treatment with 2-152a MAb, which was mediated by complement-dependent cytotoxicity (CDC). In addition, the antigen recognition domain of 2-152a MAb was responsible for the unique anti-HCV activity. These findings demonstrate the feasibility of using 2-152a MAb for antibody therapy against HCV-related HCC. In addition, surface DHCR24 on HCC cells exhibited a functional property, agonist-induced internalization. We showed that 2-152a MAb-mediated binding of a cytotoxic agent (a saponin-conjugated secondary antibody) to surface DHCR24 led to significant cytotoxicity. This suggests that surface DHCR24 on HCC cells can function as a carrier for internalization. Therefore, surface DHCR24 could be a valuable target for HCV-related HCC therapy, and 2-152a MAb appears to be useful for this targeted therapy.

Identifying the emerging human pathogen Scedosporium prolificans by using a species-specific monoclonal antibody that binds to the melanin biosynthetic enzyme tetrahydroxynaphthalene reductase.

The dematiaceous (melanized) fungus Scedosporium prolificans is an emerging and frequently fatal pathogen of immunocompromised humans and which, along with the closely related fungi Pseudallescheria boydii, Scedosporium apiospermum and S. aurantiacum in the Pseudallescheria-Scedosporium complex, is a contributing aetiology to tsunami lung and central nervous system infections in near-drowning victims who have aspirated water laden with spores. At present, the natural habitat of the fungus is largely unknown, and accurate detection methods are needed to identify environmental reservoirs of infectious propagules. In this study, we report the development of a monoclonal antibody (mAb) (CA4) specific to S. prolificans, which does not cross-react with closely related fungi in the Pseudallescheria-Scedosporium complex or with a wide range of mould and yeast species pathogenic to humans. Using genome sequencing of a soil isolate and targeted gene disruption of the CA4 antigen-encoding gene, we show that mAb CA4 binds to the melanin-biosynthetic enzyme tetrahydroxynaphthalene reductase. Enzyme-deficient mutants produce orange-brown or green-brown spore suspensions compared with the black spore suspension of the wild-type strain. Using mAb CA4 and a mAb (HG12) specific to the related fungi P. boydii, P. apiosperma, S. apiospermum and S. aurantiacum, we demonstrate how the mAbs can be used in combination with a semiselective isolation procedure to track these opportunistic pathogens in environmental samples containing mixed populations of human pathogenic fungi. Specificity of mAb CA4 was confirmed by sequencing of the internally transcribed spacer 1 (ITS1)-5.8S-ITS2 rRNA-encoding regions of fungi isolated from estuarine muds.

Monoclonal antibody 2-152a suppresses hepatitis C virus infection through betaine/GABA transporter-1.

BACKGROUND: We recently established a monoclonal antibody (2-152a MAb) that binds to 3ß-hydroxysterol-Δ24-reductase (DHCR24) by immunizing mice with cells (RzM6-LC) persistently expressing hepatitis C virus (HCV). Here, we aimed to analyze the activity of 2-152a MAb against HCV replication and explore the molecular mechanism underlying the antiviral activity. METHODS: We characterized the effects of 2-152a MAb on HCV replication and performed a microarray analysis of antibody-treated HCV replicon cells. The molecules showing a significant change after the antibody treatment were screened to examine their relationship with HCV replication. RESULTS: The antibody had antiviral activity both in vitro and in vivo (chimeric mice). In the microarray analysis, 2-152a MAb significantly suppressed the expression of betaine/GABA transporter-1 (BGT-1) in 2 HCV replicon cell lines but not in HCV-cured cells. Silencing of BGT-1 expression by small interfering RNA (siRNA) revealed significant suppression of HCV replication and infection without cytotoxicity. Further, BGT-1 expression was significantly increased in the presence of HCV (P < .05). CONCLUSIONS: Our results suggest that 2-152a MAb suppresses HCV replication and infection through BGT-1. These findings highlight important roles of BGT-1 in HCV replication and reveal a possible target for anti-HCV therapy.

Antibodies reactive to non-HLA antigens in transplant glomerulopathy.

Although T and B cell alloimmunity contribute to transplant injury, autoimmunity directed at kidney-expressed, non-HLA antigens may also participate. Because the specificity, prevalence, and importance of antibodies to non-HLA antigens in late allograft injury are poorly characterized, we used a protein microarray to compare antibody repertoires in pre- and post-transplant sera from several cohorts of patients with and without transplant glomerulopathy. Transplantation routinely induced changes in antibody repertoires, but we did not identify any de novo non-HLA antibodies common to patients with transplant glomerulopathy. The screening studies identified three reactivities present before transplantation that persisted after transplant and strongly associated with transplant glomerulopathy. ELISA confirmed that reactivity against peroxisomal-trans-2-enoyl-coA-reductase strongly associated with the development of transplant glomerulopathy in independent validation sets. In addition to providing insight into effects of transplantation on non-HLA antibody repertoires, these results suggest that pretransplant serum antibodies to peroxisomal-trans-2-enoyl-coA-reductase may predict prognosis in kidney transplantation.

Epithelial cell specificity and apotope recognition by serum autoantibodies in primary biliary cirrhosis.

UNLABELLED: A major enigma of primary biliary cirrhosis (PBC) is the selective targeting of biliary cells. Our laboratory has reported that after apoptosis, human intrahepatic biliary epithelial cells (HiBECs) translocate the E2 subunit of the pyruvate dehydrogenase complex immunologically intact into apoptotic bodies, forming an apotope. However, the cell type and specificity of this reaction has not been fully defined. To address this issue, we investigated whether the E2 subunit of the pyruvate dehydrogenase complex, the E2 subunit of the branched chain 2-oxo acid dehydrogenase complex, the E2 subunit of the oxo-glutarate dehydrogenase complex, four additional inner mitochondrial enzymes, and four nuclear antigens remain immunologically intact with respect to postapoptotic translocation in HiBECs and three additional control epithelial cells. We report that all three 2-oxo acid dehydrogenase enzymes share the ability to remain intact within the apotope of HiBECs. Interestingly, the E2 subunit of the branched chain 2-oxo acid dehydrogenase complex also remained intact in the other cell types tested. We extended the data, using sera from 95 AMA-positive and 19 AMA-negative patients with PBC and 76 controls, by testing for reactivity against the seven mitochondrial proteins studied herein and also the ability of AMA-negative sera to react with HiBEC apotopes. Sera from 3 of 95 AMA-positive sera, but none of the controls, reacted with 2,4-dienoyl coenzyme A reductase 1, an enzyme also present intact only in the HiBEC apotope, but which has not been previously associated with any autoimmune disease. Finally, the specificity of HiBEC apotope reactivity was confined to AMA-positive sera. CONCLUSION: We submit that the biliary specificity of PBC is secondary to the unique processes of biliary apoptosis.

Leflunomide inhibits PDK1/Akt pathway and induces apoptosis of human mast cells.

Mast cells release many inflammatory mediators that play an important role not only in allergic diseases but also in chronic inflammatory diseases, autoimmune diseases, and others. A lot of mast cells exist in synovium of rheumatoid arthritis, and it is known that synovitis does not occur in mast cell-deficient mice. Thus, it is thought that mast cells play a very important role in rheumatoid arthritis pathogenesis. Leflunomide is a drug used clinically in the treatment of rheumatoid arthritis. We used clinical doses of 2-cyano-3-hydroxy-N-(4-trifluoromethylphenyl)-butenamide (A77 1726), which is an active metabolite of leflunomide, and decreased the number of viable human primary mast cells in a concentration-dependent manner. This decrease was not reversed by uridine. Inhibition of pyrimidine synthesis by dihydro-orotic acid dehydrogenase inhibition, which is the primary mechanism of action of A77 1726, was not involved. A77 1726 dramatically induced apoptosis of human mast cells and inhibited the phosphorylation of Akt, an important survival signal of mast cells, in a concentration-dependent manner. Caspases 3 and 9, downstream molecules of Akt survival pathway, were also fragmented by A77 1726. In addition, it became evident for the first time that the mechanism involved in this result was the concentration-dependent inhibition of PDK1 phosphorylation, which controls the activation of Akt. These results indicate a new way of controlling mast cells and may therefore be the basis for innovative approaches to the treatment of various diseases related to mast cells.

High resolution crystal structure of PedB: a structural basis for the classification of pediocin-like immunity proteins.

BACKGROUND: Pediocin-like bacteriocins, ribosomally-synthesized antimicrobial peptides, are generally coexpressed with cognate immunity proteins in order to protect the bacteriocin-producer from its own bacteriocin. As a step for understanding the mode of action of immunity proteins, we determined the crystal structure of PedB, a pediocin-like immunity protein conferring immunity to pediocin PP-1. RESULTS: The 1.6 A crystal structure of PedB reveals that PedB consists of an antiparallel four-helix bundle with a flexible C-terminal end. PedB shows structural similarity to an immunity protein against enterocin A (EntA-im) but some disparity to an immunity protein against carnobacteriocin B2 (ImB2) in both the C-terminal conformation and the local structure constructed by alpha3, alpha4, and their connecting loop. Structure-inspired mutational studies reveal that deletion of the last seven residues of the C-terminus of PedB almost abolished its immunity activity. CONCLUSION: The fact that PedB, EntA-im, and ImB2 share a four-helix bundle structure strongly suggests the structural conservation of this motif in the pediocin-like immunity proteins. The significant difference in the core structure and the C-terminal conformation provides a structural basis for the classification of pediocin-like immunity proteins. Our mutational study using C-terminal-shortened PedBs and the investigation of primary sequence of the C-terminal region, propose that several polar or charged residues in the extreme C-terminus of PedB which is crucial for the immunity are involved in the specific recognition of pediocin PP-1.

Seladin-1 expression in rat adrenal gland: effect of adrenocorticotropic hormone treatment.

Seladin-1 (KIAA0018) gene is the seventh most highlyexpressed gene in the adult adrenal gland, along with genes coding for steroidogenic enzymes. The aim of the present study was to investigate the localization of the Seladin-1 protein in control and ACTH-treated rat adrenal glands and to verify whether Seladin-1 is involved in secretion. Immunofluorescence studies revealed that Seladin-1 was localized principally in the zona fasciculata, cytoplasm, and nucleus. Expression of Seladin-1 was increased by ACTH treatment, in vivo and in culture conditions. Subcellular fractionation offasciculata cells showed that Seladin-1 was mainly present in the nucleus, membrane, and cytoskeleton fractions and, to a lesser extent, in the cytosol. ACTH treatment decreased Seladin-1 expression in the cytosol, with a concomitant increase in the nuclear fraction. In the glomerulosa and fasciculata cells in culture, ACTH induced a relocalization of Seladin-1 into specific nuclear regions. This ACTH-induced relocalization was abrogated by the pre-treatment of cells with 75 nM U18666A (an inhibitor of Seladin-1). In addition, fasciculata cells exhibited an increase in the basal level of steroid secretion when cultured in the presence of U18666A (25 and 75 nM), although ACTH-induced secretion was decreased. In summary, the present study demonstrates that the protein expression of Seladin-1 is more abundant in fasciculata cells than in glomerulosa cells and that the ACTH treatment increases both expression and nuclear localization of the protein. Results also suggest that depending on its cellular localization, the Delta24-reductase activity of Seladin-1 may play a major role in steroid secretion in the adrenal gland.