Isolation of the Bα and Bβ mating-type loci of Schizophyllum commune

The B mating type of the basidiomycete fungus, Schizophyllum commune is determined by two, tightly linked, multi-specificity (also called multi-allelic) loci: B and B. A plasmid library was used in DNA-mediated transformation to obtain transformants that displayed B-directed development. Plasmids that conferred B1 and B1 mating-type specificities were rescued from the transformants. Fragments of DNA from each plasmid hybridized to genomic DNA from the strain used to make the plasmid library; however, they did not hybridize, or hybridized only weakly, to genomic DNA from strains with mating-type specificities different from B1 or B1. The cloned fragments are presumed to correspond to active regions of each B mating-type locus.     

Behavioral evaluation of transgenic mice with CNS expression of IFN-α by elevated plus-maze and Porsolt swim test

Chronic IFN-α treatment as an antiviral or anti-cancer therapy can lead to severe psychiatric complications, including depression and anxiety in patients. In many animal models of IFN-α-induced behavioral dysfunction, the opposite results have frequently been reported. In an attempt to overcome the limitation of pharmacological studies, IFN-α-transgenic mice with CNS-targeted expression of the IFN-α transgene were used to study depression and anxiety-like behaviors by Porsolt swim and elevated plus-maze assays, respectively. Interestingly, chronic stimulation of IFN-α signaling in mouse brains did not cause depression or anxiety as measured by these tests in comparison with wild-type littermates. This observation suggests that factors other than IFN-α may be necessary for the development of psychiatric complications following IFN-α therapy in patients.     

Rosmarinic acid interferes with influenza virus A entry and replication by decreasing GSK3β and phosphorylated AKT expression levels

BackgroundThe purpose of this study was to examine the in vivo activity of rosmarinic acid (RA) – a phytochemical with antioxidant, anti-inflammatory, and antiviral properties – against influenza virus (IAV). An antibody-based kinase array and different in vitro functional assays were also applied to identify the mechanistic underpinnings by which RA may exert its anti-IAV activity.MethodsWe initially examined the potential efficacy of RA using an in vivo mouse model. A time-of-addition assay and an antibody-based kinase array were subsequently applied to investigate mechanism-of-action targets for RA. The hemagglutination inhibition assay, neuraminidase inhibition assay, and cellular entry assay were also performed.ResultsRA increased survival and prevented body weight loss in IAV-infected mice. In vitro experiments revealed that RA inhibited different IAV viruses – including oseltamivir-resistant strains. From a mechanistic point of view, RA downregulated the GSK3β and Akt signaling pathways – which are known to facilitate IAV entry and replication into host cells.ConclusionsRA has promising preclinical efficacy against IAV, primarily by interfering with the GSK3β and Akt signaling pathways.     

Small hepatitis B surface antigen interacts with and modulates enoyl–coenzyme A hydratase expression in hepatoma cells

Enoyl–coenzyme A hydratase (ECHS1) is a key enzyme in the metabolism of fatty acids in mitochondria. We previously reported that hepatitis B surface antigen (HBsAg) interacted with ECHS1 in a yeast two-hybrid system. In the current study, we further examined their interaction by using GST pull-down and co-immunoprecipitation assays. The results confirmed that small hepatitis B surface antigen (SHBs) interacted with ECHS1. Furthermore, confocal imaging showed that SHBs and ECHS1 co-localized in HepG2 cells. To clarify the biological function of the interaction, human hepatoma cell lines that transiently and stably expressed SHBs were generated. The expression of SHBs led to a significant decrease in ECHS1 protein levels. ECHS1 protein levels were reduced to 48.44 ± 7.12 % in Huh7 cells transiently expressing SHBs, and to 54.97 ± 3.54 % in HepG2 cells stably expressing SHBs. In conclusion, our findings suggest that SHBs interacts with ECHS1 and regulates ECHS1 protein levels in hepatoma cells.     

Production and expression of RANTES (CCL5) by human disc cells and modulation by IL-1-β and TNF-α in 3D culture

Chemokines act as important secondary inflammatory mediators which are released by cells in response to a variety of stimuli. Chemokines bind to cell surface receptors and act as second-order cytokines with specialized functions in inflammation. The role of RANTES (Regulated upon Activation, Normal T-cell Expressed, and Secreted) (also called CCL5 (chemokine (C–C motif) ligand 5)) has received little attention to date in disc tissue. Microarray analyses of lumbar disc annulus tissue revealed that RANTES expression was significantly upregulated in more degenerated Thompson grades IV and V discs compared to expression levels in grades I, II and III discs (p = 0.032). Immunolocalization confirmed the presence of RANTES in the annulus and nucleus of the disc, and localized the RANTES receptors CCR1, CCR3 and CCR5 to cells in the disc. In vitro studies with IL-1-β and TNF-α challenges, both proinflammatory cytokines resulted in elevated levels of RANTES in conditioned media (p < 0.01); TNF-α exposure, however, produced significantly greater levels than did IL-1alpha (p < 0.0001), suggesting a differential regulation by TNF-α. Local production of RANTES in vivo by annulus and nucleus cells, and in vitro induction of RANTES by proinflammatory cytokines suggest that disc cells are primary effector cells as well as target cells, and thus can mediate physiological immune-related processes during disc degeneration by both autocrine and paracrine signaling.     

Genomic and sequence variants of protein kinase A regulatory subunit type 1β (PRKAR1B) in patients with adrenocortical disease and Cushing syndrome

PurposeProtein kinase A (PKA) subunit defects (in PRKAR1A and PRKACA) are known to contribute to adrenal tumor pathogenesis. We studied the PRKAR1B gene for any genetic changes in bilateral adrenocortical hyperplasia (BAH) and cortisol-producing adrenal adenomas (CPA).MethodsExome sequencing and PRKAR1B copy-number variant (CNV) analysis were performed in 74 patients with BAH and 21 with CPA. PKA activity was studied in tumors with defects; sequence variants were investigated in vitro.ResultsThree PRKAR1B germline variants (p.I40V, p.A67V, p.A300T) were identified among 74 patients with BAH. PRKAR1B copy-number gains (CNG) were found in 3 of 21 CPAs, one in a tumor carrying a somatic PRKACA “hotspot” pathogenic variant p.L206R. CPAs bearing PRKAR1B CNGs showed higher PRKAR1B messenger RNA (mRNA) levels and reduced PKA activity. Baseline PKA activity was also decreased for p.A67V and p.A300T in vitro, and mutant PRKAR1β bound PRKACα in fluorescence resonance energy transfer (FRET) recordings of cotransfected HEK293 cells stronger than normal.ConclusionPRKAR1B is yet another PKA subunit that may potentially contribute to adrenal tumor formation. Its involvement in adrenocortical disease may be different from that of other subunits, because PRKAR1B variants and PRKAR1B CNGs were associated with decreased (rather than increased) overall PKA activity in vitro.     

Discoordinate expression of IL-1α and IL-1β in interfacial membranes of failed joint prostheses

Bone resorption following either cemented or uncemented total hip replacement has been implicated as an important etiologic factor in aseptic loosening of prostheses, the most frequent cause of clinical failure. Interleukin-1 (IL-1), collagenase and prostaglandin E₂ are considered to play key roles in pathological bone resorption. We have measured the actual levels and quantified the genes coding for several cytokines [IL-1, IL-1, IL-4, IL-6, platelet-derived growth factors (PDGF), transforming growth factor- (TGF) and tumor necrosis factor- (TNF)] in interfacial membranes obtained from cemented or uncemented loosened joint replacements. IL-1, IL-6 and TNF were barely detectable in the interfacial membranes either at protein or mRNA levels, while IL-1 and TGF were found to be expressed at the highest levels in freshly isolated tissues. However, the expression of IL-1 increased 10–1000-fold either in isolated cells or explant cultures of interfacial membranes within 24 h. The expression of other cytokines, measured directly in tissue or cells, did not suggest a discoordinate expression of bone-resorbing cellular mediators.     

Correlation of serum hepatitis B surface antigen level with response to entecavir in naïve patients with chronic hepatitis B

Recent studies have suggested that quantifying the serum HBsAg levels can predict the response to pegylated interferon. We aimed to determine the change in serum HBsAg levels during entecavir (ETV) treatment and the correlation with treatment response in chronic HBeAg‐positive and HBeAg‐negative hepatitis B patients. Serial HBsAg levels were measured using the Architect assay (Abbott Laboratories, Abbott Park, IL) in sera from 101 treatment‐naive chronic hepatitis B (CHB) patients receiving ETV. During treatment, in HBeAg‐positive patients, the mean HBsAg level was 3.51, 3.22, 3.34, 3.36, and 3.40 log₁₀ IU/ml at baseline, 3, 6, 12, and 24 months, respectively, and there was no significant change compared with the baseline level, except the decline at 3 months (P = 0.009). In HBeAg‐negative patients, the mean level of serum HBsAg showed increase with 3.06, 3.09, 3.20, 3.26, and 3.27 log₁₀ IU/ml at baseline, 3, 6, 12, and 24 months of treatment, respectively. In HBeAg‐positive patients, HBV‐DNA negativity (<2,000 copies/ml; P = 0.010) and HBsAg level <3,000 IU/ml (P = 0.026) at 3 months were independent predictors of HBeAg loss/seroconversion at 12 months. After 24 months of treatment, the HBsAg levels at baseline (P = 0.046) was an independent factor of HBeAg loss/seroconversion. In HBeAg‐negative patients, undetectable HBV DNA at 6 months was an independent factor predicting undetectable HBV DNA after 12 months of therapy. The level of serum HBsAg before and during therapy was a good predictor of HBeAg loss/seroconversion in naïve HBeAg‐positive CHB patients receiving entecavir. J. Med. Virol. 83:1178–1186, 2011. © 2011 Wiley‐Liss, Inc.     

Attenuated expression of interferon-β and interferon-λ1 by human alternatively activated macrophages

Macrophages can be polarized into classically (CAM) or alternatively (AAM) activated macrophages with IFN-γ or IL-4, respectively. CAM are associated with type 1 immune responses and are implicated in autoimmunity; AAM are associated with type 2 responses and are implicated in allergic diseases. An impediment in investigating macrophage biology using primary human monocyte derived macrophages is the wide inter-donor heterogeneity and the limited quantity of cells that survive in vitro polarization. To overcome this impediment, we established a protocol to generate CAM and AAM cultures derived from the THP-1 human promonocytic cell line. In this report, we demonstrate that THP-CAM and -AAM express gene and protein markers that define their primary human monocyte derived counterparts, such as IL-1β, CXCL10, and CXCL11 for CAM, and MRC1, IL-4 and CCL22 for AAM. In addition, we demonstrate that STAT6 is selectively activated in THP-AAM which, upon LPS stimulation, have an attenuated or delayed expression of IFN-β, IFN-λ1, and IFN α/β pathway genes compared to their CAM counterparts. Taken together, these findings may help further investigate human diseases associated with the alternatively activated macrophage phenotype using this reproducible in vitro macrophage model.     

Nonstructural protein 1α subunit-based inhibition of NF-κB activation and suppression of interferon-β production by porcine reproductive and respiratory syndrome virus

Induction of type I interferon (IFN-α/β) is an early antiviral response of the host, and porcine reproductive and respiratory syndrome virus (PRRSV) has been reported to downregulate the IFN response during infection in cells and pigs. We report that the PRRSV nonstructural protein 1α (Nsp1α) subunit of Nsp1 is a nuclear-cytoplasmic protein distributed to the nucleus and contains a strong suppressive activity for IFN-β production that is mediated through the retinoic acid-inducible gene I (RIG-I) signaling pathway. Nsp1α suppressed the activation of nuclear factor (NF)-κB when stimulated with dsRNA or tumor necrosis factor (TNF)-α, and NF-κB suppression was RIG-I-dependent. The suppression of NF-κB activation was associated with the poor production of IFN-β during PRRSV infection. The C-terminal 14 amino acids of the Nsp1α subunit were critical in maintaining immunosuppressive activity of Nsp1α for both IFN-β and NF-κB, suggesting that the newly identified zinc finger configuration comprising of Met180 may be crucial for inhibitory activities. Nsp1α inhibited IκB phosphorylation and as a consequence NF-κB translocation to the nucleus was blocked, leading to the inhibition of NF-κB stimulated gene expression. Our results suggest that PRRSV Nsp1α is a multifunctional nuclear protein participating in the modulation of the host IFN system.     

Multitasking of Ig-α and Ig-β to Regulate B Cell Antigen Receptor Function

Since their discovery as signaling subunits of the B cell antigen receptor (BCR), Ig-α and Ig-β are discussed to serve either a redundant or distinct function for B cell development, maintenance, and activation. Dependent upon the experimental system that has been used to address this issue, evidence could be provided to support both possibilities. Only recently has it become clear that Ig-α and Ig-β possess a unique signaling identity but that both together are required to orchestrate proper B cell function in vivo. Here we discuss some of the underlying mechanisms that may involve direct coupling to discrete subsets of BCR effector proteins, such as protein tyrosine kinases or the intracellular adaptor SLP-65/BLNK.     

Impaired expression of Act1mRNA in B cells of patients with Sjögren's syndrome

Sj?gren's syndrome (SS) is a systemic autoimmune disorder characterized by profound lymphocytic infiltration into the lacrimal and salivary glands, thereby diminished secretory function. B cell hyper-activation is a predominant feature of SS related to hypergammaglobulinemia and production of autoantibodies. The adaptor molecule NF-kB activator 1 (Act1) plays an important role in the homeostasis of B cells by attenuating CD40 and B cell-activating factor belonging to the tumor necrosis factor family receptor (BAFFR) signaling. Act1-deficient mice develop autoimmune manifestations similar to SS, which are hypergammaglobulinemia, high levels of anti-SSA and anti-SSB autoantibodies. In this study, to investigate the role of Act1 in the pathogenesis of SS, we examined Act1mRNA expressions in B cells from patients with SS and discussed the association of Act1 with parameters and clinical manifestations of SS. We showed the low level of Act1mRNA expression in patients with SS and reciprocal association of Act1 with serum IgG level. Diminished Act1mRNA expression in SS may be associated with B cell hyperactivity and elevated immunoglobulin production in SS by uncontrolled B cell activation signal through CD40 and BAFFR.     

Effects of interleukin-1β and tumor necrosis factor-α on osteoblastic expression of osteocalcin and mineralized extracellular matrix in vitro

Osteoblasts play a pivotal role during the bioresponse of bone to agents that stimulate bone resorption and/or inhibit bone formation including hormones, polypeptide growth factors, and cytokines. We examined the cytokines interleukin-1-beta (IL-1) and tumor necrosis factor-alpha (TNF-) for their effects on osteoblastic proliferation and development and expression of alkaline phosphatase and the osteoblast-specific protein osteocalcin in a mineralizing environment. Primary rat osteoblast-like cells (ROB) and osteoblastic cell lines derived from rat (ROS 17/2.8) and human (MG-63) osteosarcomas were studied. IL-1 and TNF- were chosen because of their critical importance during the host response to local inflammatory stimuli. Qualitatively similar two- to threefold inhibition of osteocalcin synthesis by IL-1 and TNF- were observed in all three postconfluent bone-forming model systems. Because of the readily measurable concentrations of osteocalcin produced in our culture protocol, it was not necessary to enhance osteoblastic synthesis of osteocalcin by supplementation with 1,25(OH)₂-vitamin D₃, a treatment which exerts pleiotropic effects on osteoblasts. Under the constraints of our protocol, where alkaline phosphatase and mineralization were already elevated at the 14-day onset of treatment, neither of these phenotypic properties was sensitive to a three-day cytokine exposure. Differences were noted in proliferation, where only TNF- stimulated DNA synthesis in ROB cells, while both cytokines stimulated MG-63 cells. IL-1 and TNF- failed to alter ROS 17/2.8 DNA synthesis except at the highest doses (25 pM IL-1 and l nM TNF-) where inhibition was observed. These results further support the view that cytokine-mediated osteoblastic regulation can be relatively selective.     

Tumor necrosis factor-α induces expression and release of interleukin-6 by human urothelial cells

Objective  

We examined the effects of tumor necrosis factor-α (TNF-α) on expression and release of interleukin-6 (IL-6) by human urothelial cells (HUCs) and investigated whether the effects of TNF-α are mediated by mitogen-activated protein kinase (MAPK) pathways.     

ALK-negative anaplastic large cell lymphoma with “Hodgkin-like” cytomorphology and nuclear expression of PAX5

Anaplastic lymphoma kinase negative systemic anaplastic large cell lymphoma (ALK-ALCL) is a CD30+ T-cell malignant lymphoma which may involve both lymph nodes and extranodal tissues, showing important clinical differences from ALK-positive ALCL (ALK + ALCL). ALK- ALCL is considered a specific entity by the 2016 World Health Organization (WHO) classification of hematolymphoid neoplasms.We describe an exceptional case of ALK- ALCL with a striking “Hodgkin-like” cytomorphology and a very uncommon nuclear expression of PAX5.     

PPM1B and P-IKKβ expression levels correlated inversely with rat gastrocnemius atrophy after denervation

Activated inhibitor of nuclear factor-κB kinase β (IKKβ) is necessary and sufficient for denervated skeletal muscle atrophy. Although several studies have shown that Mg²⁺/Mn²⁺-dependent protein phosphatase 1B (PPM1B) inactivated IKKβ, few studies have investigated the role of PPM1B in denervated skeletal muscle. In this study, we aim to explore the expression and significance of PPM1B and phosphorylated IKKβ (P-IKKβ) during atrophy of the denervated gastrocnemius. Thirty young adult female Wistar rats were subjected to right sciatic nerve transection and were sacrificed at 0 (control), 2, 7, 14, and 28 days after denervation surgery. The gastrocnemius was removed from both the denervated and the contralateral limb. The muscle wet weight ratio was calculated as the ratio of the wet weight of the denervated gastrocnemius to that of the contralateral gastrocnemius. RT-PCR and Western blot analysis showed that mRNA and protein levels of PPM1B were significantly lower than those of the control group at different times after the initiation of denervation, while P-IKKβ showed the opposite trends. PPM1B protein expression persistently decreased while P-IKKβ expression persistently increased for 28 days after denervation. PPM1B expression correlated negatively with P-IKKβ expression by the Spearman test, whereas decreasing PPM1B expression correlated positively with the muscle wet weight ratio. The expression levels of PPM1B and P-IKKβ were closely associated with atrophy in skeletal denervated muscle. These results suggest that PPM1B and P-IKKβ could be markers in skeletal muscle atrophy.     

Correlations of TOP2A gene aberrations and expression of topoisomerase IIα protein and TOP2A mRNA expression in primary breast cancer: a retrospective study of 86 cases using fluorescence in situ hybridization and immunohistochemistry

Our aim in this study was to assess the status of TOP2A gene aberrations (no change/amplification or deletion) and its correlations with topoisomerase IIα (Topo IIα) protein and TOP2A mRNA expression, respectively. TOP2A amplification, Topo IIα protein expression and TOP2A mRNA expression were assessed using samples of 86 cases of breast cancer by fluorescence in fluorescence in situ hybridization, quantitative real-time polymerase chain reaction and immunohistochemistry, respectively. Twenty two (22.57%) had amplification/deletion of TOP2A gene. Twenty eight (32.56%) tumor samples were 17q polysomy or monosomy. Topo IIα protein was expressed in 57 cases (66.27%, 57/86): 22 cases (38.62%, 22/57) and 35 cases (61.40%, 35/57) had amplification/deletion and no change of TOP2A gene, respectively. These three groups showed significant differences by one-way analysis of variance (P < 0.001). The average Ct values of TOP2A mRNA expression in the tumors with deletion, amplification and no change of TOP2A gene were 27.00, 27.33 and 31.66, respectively. We demonstrated that the TOP2A gene was amplified or deleted in breast cancer, with a significant correlation with high expressions of Topo IIα protein and TOP2A mRNA expression. Ki-67 expression index (mean = 14.9) decreased significantly in cases wherein TOP2A gene had no change and Her2/neu protein expression was weakly positive (0-1+, P < 0.001).     

IL-36α induces maturation of Th1-inducing human MDDC and synergises with IFN-γ to induce high surface expression of CD14 and CD11c

We show that IL-36α induced maturation of human MDDCs and stimulated differentiation of IFN-γ producing (Type 1) CD3+ lymphocytes but was not as effective as IL-36β in doing so. For the first time, we also show that IL-36α induced expression of CD14 by MDDCs and this was highly potentiated by co-cultured with IFN-γ. In contrast, lipopolysaccharide (LPS) did not increase CD14 expression by MDDCs, suggesting that if MDDCs represent a physiologically relevant population in vivo, they need to be stimulated by relevant inflammatory cytokines prior to CD14 expression and detection of LPS, expressed by Gram negative bacteria. IFN-γ synergised with IL-36α to restore the high levels of CD11c expression by MDDCs, which was reduced by culture with these cytokines in isolation. IL-36α/IFN-γ synergy also correlated with increased binding of the opsonic complement protein (iC3b) to MDDCs. However although IL-36α increased the phagocytic capacity of MDDCs for Salmonella Typhimurium 4/74 this was not synergistically increased by IFN-γ (P > 0.05).In conclusion we report the hitherto unknown effects of IL-36α on the innate cell function of human MDDCs.