Porphyromonas gingivalis Cell Wall Components Induce Programmed Death Ligand 1 (PD-L1) Expression on Human Oral Carcinoma Cells by a Receptor-Interacting Protein Kinase 2 (RIP2)-Dependent Mechanism.

Programmed death-ligand 1 (PD-L1/B7-H1) serves as a cosignaling molecule in cell-mediated immune responses and contributes to chronicity of inflammation and the escape of tumor cells from immunosurveillance. Here, we investigated the molecular mechanisms leading to PD-L1 upregulation in human oral carcinoma cells and in primary human gingival keratinocytes in response to infection with Porphyromonas gingivalis (P. gingivalis), a keystone pathogen for the development of periodontitis. The bacterial cell wall component peptidoglycan uses bacterial outer membrane vesicles to be taken up by cells. Internalized peptidoglycan triggers cytosolic receptors to induce PD-L1 expression in a myeloid differentiation primary response 88 (Myd88)-independent and receptor-interacting serine/threonine-protein kinase 2 (RIP2)-dependent fashion. Interference with the kinase activity of RIP2 or mitogen-activated protein (MAP) kinases interferes with inducible PD-L1 expression.

Ischemic stroke subtype is associated with outcome in thrombolyzed patients.

OBJECTIVES: The impact of ischemic stroke subtype on clinical outcome in patients treated with intravenous tissue-type plasminogen activator (IV-tPA) is sparsely examined. We studied the association between stroke subtype and clinical outcome in magnetic resonance imaging (MRI)-evaluated patients treated with IV-tPA. MATERIAL AND METHODS: We conducted a single-center retrospective analysis of MRI-selected stroke patients treated with IV-tPA between 2004 and 2010. The Trial of ORG 10172 in Acute Stroke Treatment criteria were used to establish the stroke subtype by 3 months. The outcomes of interest were a 3-month modified Rankin Scale score of 0-1 (favorable outcome), and early neurological improvement defined as complete remission of neurological deficit or improvement of ≥4 on the National Institute of Health Stroke Scale at 24 h. The outcomes among stroke subtypes were compared with multivariable logistic regression. RESULTS: Among 557 patients, 202 (36%) had large vessel disease (LVD), 153 (27%) cardioembolic stroke (CE), 109 (20%) small vessel disease, and 93 (17%) were of other or undetermined etiology. Early neurological improvement was present in 313 (56.4%) patients, and 361 (64.8%) patients achieved a favorable outcome. Early neurological improvement and favorable outcome were more likely in CE patients compared with LVD patients (odds ratio (OR), 2.1 (95% confidence interval, 1.4-3.3), and 2.0 (95% confidence interval, 1.2-3.3), respectively). CONCLUSIONS: Cardioembolic stroke patients were more likely to achieve early neurological improvement and favorable outcome compared with LVD stroke following MRI-based IV-tPA treatment. This finding may reflect a difference in the effect of IV-tPA among stroke subtypes.

Sex reversal by loss of the C-terminal transactivation domain of human SOX9.

Haploinsufficiency for SOX9 has recently been identified as the cause for both campomelic dysplasia (CD), a human skeletal malformation syndrome, and the associated autosomal XY sex reversal. SOX9 contains a putative DNA-binding motif known as the high-mobility group (HMG) domain characterizing a whole class of transcription factors. We show in cell transfection experiments that SOX9 can transactivate transcription from a reporter plasmid through the motif AACAAAG, a sequence recognized by other HMG domain transcription factors. By fusing all or part of SOX9 to the DNA-binding domain of yeast GAL4, the transactivating function was mapped to a transcription activation (TA) domain at the C terminus of SOX9. This non-acidic TA domain is evolutionarily conserved and rich in proline, glutamine and serine. With one exception, all SOX9 nonsense and frame shift mutations described so far in CD/sex reversal patients lead to truncation of the TA domain, suggesting that impairment of gonadal and skeletal development in these cases results, at least in part, from loss of transactivation of genes downstream of SOX9.

Improved intraportal islet transplantation outcome by systemic IKK-beta inhibition: NF-κB activity in pancreatic islets depends on oxygen availability.

Intraportal islet transplantation suffers from low efficiency caused by substantial islet mass loss after transplantation. How this process is regulated is still unclear. Here, we show that NF-κB activation was detectable in islet grafts shortly after transplantation of porcine islets to diabetic NMRI nu/nu mice, and systemic NF-κB inhibition in transplanted animals significantly prolonged islet graft survival. Proinflammatory cytokines alone did not cause evident cell death in pancreatic islet within 24 h, while the combination of cytokines with hypoxia resulted in a strong induction of cell death that could be blocked dose-dependently by a selective IKK-ß inhibitor. Under hypoxia, NF-κB activity impaired expression of antiapoptotic gene BCL-xL, c-FLIP and survivin. NF-κB activation in isolated islets was reduced by hypoxia in a time-dependent manner, accordingly, NF-κB activation in transplanted islets diminished by time. Our data indicate that, while NF-κB has an antiapoptotic role under normoxia, low oxygen conditions decrease its activity and transform it to a proapoptotic transcription factor in pancreatic islets. We conclude that NF-κB inhibition represents a potential strategy to improve islet transplantation efficiency.

Proteins controlling the nuclear uptake of NF-kappa B, Rel and dorsal.

The two DNA-binding subunits of the transcription factor NF-kappa B, the products of the rel oncogene family and the product of the developmental control gene dorsal of Drosophila are homologous within a 300 amino acid region. This sequence represents a novel DNA-binding and dimerization domain. The access of the NF-kappa B/Rel/dorsal (NRD) transcription factor family to the cell nucleus is regulated. There is now evidence that functionally and structurally related accessory proteins of NF-kappa B, Rel and dorsal control the nuclear entry as well as DNA-binding activity of the transcription factors. This review summarizes current knowledge about the nuclear-uptake regulatory proteins (NURPs) I kappa B-alpha, I kappa B-beta/pp40 and cactus.

I kappa B-independent control of NF-kappa B activity by modulatory phosphorylations.

Activation of the transcription factor nuclear factor kappa B (NF-kappa B) requires its release from inhibitor of NF-kappa B (I kappa B) proteins in the cytoplasm. Much work has focussed on the identification of pathways regulating this cytosolic rate-limiting step of NF-kappa B activation. However, there is increasing evidence for another complex level of NF-kappa B activation, which involves modulatory phosphorylations of the DNA-binding subunits. These phosphorylations can control several functions of NF-kappa B, including DNA binding and transactivation properties, as well as interactions between the transcription factor and regulatory proteins. Although their overall impact on NF-kappa B function has yet to be determined, modifications of this factor will very probably provide a mechanism to fine tune NF-kappa B function.

Olecranon fractures in children and adolescents: outcomes based on fracture fixation.

PURPOSE: Open reduction and internal fixation with a tension band construct is the standard treatment for displaced transverse intra-articular olecranon fractures. The purpose of this study is to describe the outcomes of tension band fixation of olecranon fractures in children, specifically assessing the need for revision fixation and hardware removal. METHODS: Patients less than 18 years of age diagnosed with a displaced transverse intra-articular olecranon fracture and treated with tension band fixation between 2008 and 2017 were retrospectively enrolled. Operative treatment was with tension band wire (TBW) or tension band suture (TBS) constructs. RESULTS: A total of 46 patients, 36 male and ten female with a mean age of 12.3 years (6 to 17), were included. Surgical fixation was with TBW in 17 patients and TBS in 29 patients. Revision fixation due to failure and fracture displacement was required in 6% of the TBW group and 14% of the TBS group (p = 0.19). The patients who required revision fixation in the TBS group were older (14.7 years versus 11.6 years, p = 0.05) and heavier (70.5 kg versus 48.5 kg, p = 0.05) than those in the same group who did not require revision fixation. CONCLUSION: Paediatric olecranon fractures treated with TBW or TBS fixation unite in the majority of patients with similar need for hardware removal due to prominence and/or pain between fixation techniques. In a select group of older patients weighing greater than 50 kg, TBS constructs demonstrate increased failure rates, requiring revision fixation, and should be avoided in this population group. LEVEL OF EVIDENCE: IV.

Controlling NF-kappaB activation in T cells by costimulatory receptors.

Full and productive activation of T lymphocytes relies on the simultaneous delivery of T cell receptor (TCR)- and coreceptor-derived signals. In naïve T cells engagement of the TCR alone causes anergy, while TCR triggering of preactivated T cells results in activation-induced cell death. Costimulatory signals are prominently mirrored by the activation of NF-kappaB, which needs input from the TCR as well as from coreceptors in order to be fully activated and to fulfil its crucial function in the immune response. Coreceptor-generated signals tightly control the duration and amplitude of the NF-kappaB response. The activation of IkappaB kinase (IKK) complex at the contact zone between a T cell and an antigen-presenting cell offers the unique opportunity to study the spatial organization of IKK activation. Recent studies indicate that coreceptor pathways influence the threshold activities of many signalling mediators and thus act on multiple layers of the NF-kappaB pathway.

The dual effect of adenovirus type 5 E1A 13S protein on NF-kappaB activation is antagonized by E1B 19K.

The genomes of human adenoviruses encode several regulatory proteins, including the two differentially spliced gene products E1A and E1B. Here, we show that the 13S but not the 12S splice variant of E1A of adenovirus type 5 can activate the human transcription factor NF-kappaB in a bimodal fashion. One mode is the activation of NF-kappaB containing the p65 subunit from the cytoplasmic NF-kappaB-IkappaB complex. This activation required reactive oxygen intermediates and the phosphorylation of IkappaBalpha at serines 32 and 36, followed by IkappaBalpha degradation and the nuclear uptake of NF-kappaB. In addition, 13S E1A stimulated the transcriptional activity of the C-terminal 80 amino acids of p65 at a core promoter with either a TATA box or an initiator (INR) element. The C-terminal 80 amino acids of p65 were found to associate with E1A in vitro. The activation of NF-kappaB-dependent reporter gene transcription by E1A was potently suppressed upon coexpression of the E1B 19-kDa protein (19K). E1B 19K prevented both the activation of NF-kappaB and the E1A-mediated transcriptional enhancement of p65. These inhibitory effects were not found for the 55-kDa splice variant of the E1B protein. We suggest that the inductive effect of E1A 13S on the host factor NF-kappaB, whose activation is important for the transcription of various adenovirus genes, must be counteracted by the suppressive effect of E1B 19K so that the adenovirus-infected cell can escape the immune-stimulatory and apoptotic effects of NF-kappaB.

Mixed-lineage kinase 3 delivers CD3/CD28-derived signals into the IkappaB kinase complex.

The phosphorylation of IkappaB by the multiprotein IkappaB kinase complex (IKC) precedes the activation of transcription factor NF-kappaB, a key regulator of the inflammatory response. Here we identified the mixed-lineage group kinase 3 (MLK3) as an activator of NF-kappaB. Expression of the wild-type form of this mitogen-activated protein kinase kinase kinase (MAPKKK) induced nuclear immigration, DNA binding, and transcriptional activity of NF-kappaB. MLK3 directly phosphorylated and thus activated IkappaB kinase alpha (IKKalpha) and IKKbeta, revealing its function as an IkappaB kinase kinase (IKKK). MLK3 cooperated with the other two IKKKs, MEKK1 and NF-kappaB-inducing kinase, in the induction of IKK activity. MLK3 bound to components of the IKC in vivo. This protein-protein interaction was dependent on the central leucine zipper region of MLK3. A kinase-deficient version of MLK3 strongly impaired NF-kappaB-dependent transcription induced by T-cell costimulation but not in response to tumor necrosis factor alpha or interleukin-1. Accordingly, endogenous MLK3 was phosphorylated and activated by T-cell costimulation but not by treatment of cells with tumor necrosis factor alpha or interleukin-1. A dominant negative version of MLK3 inhibited NF-kappaB- and CD28RE/AP-dependent transcription elicited by the Rho family GTPases Rac and Cdc42, thereby providing a novel link between these GTPases and the IKC.

Caspase-dependent cleavage and inactivation of the Vav1 proto-oncogene product during apoptosis prevents IL-2 transcription.

Here we identify the hematopoietic proto-oncogene Vav1 as a caspase substrate during apoptosis in lymphoid cells. Cleavage of Vav1 is prevented by the caspase inhibitors zDEVD and zVAD as well as by expression of CrmA. Vav1 is cleaved in vivo at the evolutionary conserved caspase consensus cleavage site DLYD161C, generating the carboxy-terminal cleavage product Vav1p76 of intermediate stability. In vitro caspase assays reveal cleavage of Vav1 at position 161 either by apoptotic cell lysates or by recombinant caspase-3. Mutation of Asp 161 to Ala leads to the usage of the adjacent alternative cleavage sequence DQID150D. Mutation of both cleavage sites at position 150 and 161 protects Vav1 from caspase-mediated proteolysis in vitro and in vivo. The cleavage product Vav1p76 is capable of activating JNK in T-cells, but fails to induce the phosphorylation of p38/HOG1. Vav1p76 displays a diminished capacity to activate the transcription factors NF-AT, AP-1 and NF-kappaB, and thus completely fails to activate IL-2 transcription. Since Vav1 is essential for IL-2 production and plays a central role for cytoskeletal reorganization, its proteolytic inactivation during apoptosis affects multiple downstream targets.

Repression of NF-kappaB impairs HeLa cell proliferation by functional interference with cell cycle checkpoint regulators.

NF-kappaB is an inducible transcription factor, which is regulated by interaction with inhibitory IkappaB proteins. Previous studies linked the activity of NF-kappaB to the proliferative state of the cell. Here we have analysed the function of NF-kappaB in the cell cycle. Inhibition of NF-kappaB in HeLa cells by stable overexpression of a transdominant negative IkappaB-alpha protein reduced cell growth. A kinetic analysis of the cell cycle revealed a retarded G1/S transition. The IkappaB-alpha overexpressing cell clones showed a decreased percentage of cells in the S phase and an impaired incorporation of bromodeoxyuridine (BrdU). The amounts of cyclins A, B1, D1, D3, and E were unchanged, but the G1-specific proteins cyclin D2 and cdk2 were strongly elevated in the IkappaB-alpha overexpressing cell clones. These cell clones also displayed an increase in cyclin D1-dependent kinase activity, pointing to a cell cycle arrest at the late G1 phase. IkappaB-alpha overexpression crosstalked to cell cycle checkpoints via a reduction of transcription factor p53 and elevation of p21WAF. Surprisingly, the IkappaB-alpha overexpressing cells showed an enrichment of c-Myc in the nucleoli, although the total amount of c-Myc protein was unchanged. These experiments identify an important contribution of the NF-kappaB/IkappaB system for the growth of HeLa cells.

Hydrogen peroxide-induced apoptosis is CD95-independent, requires the release of mitochondria-derived reactive oxygen species and the activation of NF-kappaB.

Reactive oxygen species (ROS) play an important role in cell death induced by many different stimuli. This study shows that hydrogen peroxide-induced apoptosis in T-cells did not require tyrosine kinase p561ck, phosphatase CD45, the CD95 receptor and its associated Caspase-8. H2O2-triggered cell death led to the induced cleavage and activation of Caspase-3. Hydrogen peroxide-treatment of T-cells resulted in the formation of mitochondrial permeability transition pores, a rapid decrease of the mitochondrial transmembrane potential delta psi(m) and the release of Cytochrome C. Inhibition of the mitochondrial permeability transition by bongkrekic acid (BA), or interference with the mitochondrial electron transport system by rotenone or menadione prevented the cytotoxic effect of H2O2. Antimycin A, a mitochondrial inhibitor that increases the release of mitochondrial ROS (MiROS), enhanced apoptosis. Overexpression of Bcl-2 and the viral anti-apoptotic proteins BHRF-1 and E1B 19K counteracted H2O2-induced apoptosis. Pharmacological and genetic inhibition of transcription factor NF-kappaB protected cells from hydrogen peroxide-elicited cell death. This detrimental effect of NF-kappaB mediating hydrogen peroxide-induced cell death presumably relies on the induced expression of death effector genes such as p53, which was NF-kappaB-dependently upregulated in the presence of H2O2.

Inhibition of tyrosine phosphatases induces apoptosis independent from the CD95 system.

The inhibition of protein tyrosine phosphatases by pervanadate, a potent activator of B- and T-cells through the induction of tyrosine phosphorylation and downstream signaling events in different activation cascades, efficiently induced apoptosis in lymphoid cell lines. Pervanadate-elicited apoptosis could be blocked by the tyrosine kinase inhibitor herbimycin A. This apoptotic process involved the activation of caspases 3, 8 and 9, the induction of mitochondrial permeability transition, the release of cytochrome C and the fragmentation of chromosomal DNA. T-cells lacking the CD95 receptor or caspase-8 and T-cells stably overexpressing a transdominant negative form of the adaptor protein FADD were still susceptible to pervanadate-induced apoptosis, excluding the involvement of the CD95 system or other FADD-dependent death receptors. The apoptotic program initiated by the inhibition of tyrosine phosphatases did not require the presence of the tyrosine kinase p56lck or phosphatase CD45, whereas Bcl-2 overexpression protected T-cells from pervanadate-induced cytochrome C release, caspase-8 cleavage and apoptosis.

Relationship of ECMO duration with outcomes after pediatric cardiac surgery: a multi-institutional analysis.

BACKGROUND: There are very sparse data on the outcomes of children receiving prolonged extracorporeal membrane oxygenation (ECMO) after cardiac surgery. This study was aimed to evaluate the association of ECMO duration with outcomes in children undergoing surgery for congenital heart disease using the Pediatric Health Information System (PHIS) database. METHODS: Patients aged ≤18 years receiving ECMO after pediatric cardiac surgery (with or without cardiopulmonary bypass) at a PHIS-participating hospital (2004-2013) were included. De-identified data obtained from retrospective, observational dataset included demographic information, baseline characteristics, pre-ECMO risk factors, operation details, patient diagnoses, and center data. Outcomes evaluated included in-hospital mortality, length of mechanical ventilation, length of ICU stay, length of hospital stay, and hospital charges. Cox proportional hazards models were fitted to study the probability of study outcomes as a function of ECMO duration. RESULTS: Nine hundred ninety-eight patients from 37 hospitals qualified for inclusion. The median duration of ECMO run was 4 days (IQR: 1.7). After adjusting for patient and center characteristics, there was 12% increase in the odds of mortality for every 24 hours increase in ECMO duration (OR: 1.12, 95% CI: 1.07-1.18, P<0.001). Patients receiving longer duration of ECMO were associated with longer length of mechanical ventilation, longer length of ICU stay, longer length of hospital stay, and higher hospital charges. CONCLUSION: Data from this large multicenter database suggest that longer duration of ECMO support after pediatric cardiac surgery is associated with worsening outcomes.

The Drosophila proteins Pelle and Tube induce JNK/AP-1 activity in mammalian cells.

The mammalian interleukin-1 (IL-1) signal transduction pathways display remarkable homology to the Toll signaling cascade in Drosophila. To address the question whether members of the Drosophila Toll pathway are functional in mammalian cells, inactive and constitutively active versions of the protein kinase Pelle and its regulator Tube were expressed in HeLa cells and tested for their impact on IL-1-dependent signaling events. The Drosophila proteins failed to induce the IL-1-responsive transcription factor, nuclear factor-kappaB, but selectively activated the IL-1-regulated kinase, c-Jun N-terminal kinase (JNK), thus resulting in elevated AP-1 activity. Activation of JNK/AP-1 activity was seen upon expression of a Pelle mutant lacking its C-terminal half or by a membrane-bound and multimerised Tube protein, showing the functionality of the Drosophila proteins in mammalian cells.

Sesquiterpene lactone containing Mexican Indian medicinal plants and pure sesquiterpene lactones as potent inhibitors of transcription factor NF-kappaB.

The potential inhibitory effect of 54 Mexican Indian medicinal plants on the activation of transcription factor NF-kappaB was studied. Band-shift experiments identified the ethanolic leaf extracts of Artemisia ludoviciana ssp. mexicana, Calea zacatechichi, and Polymnia maculata (all rich in sesquiterpene lactones) as inhibitors of NF-kappaB down to a concentration of 25 microg/ml. The sesquiterpene lactones isohelenin and parthenolide prevented NF-kappaB activation completely as low as 5 microM. Treatment of HeLa cells with leaf extract of A. ludoviciana ssp. mexicana, isohelenin and parthenolide prevented the induction of transcription on the IL-6 promoter. These experiments identify the eudesmanolide and germacranolide type of sesquiterpene lactones as potent non-antioxidant inhibitors of NF-kappaB. All plants active in the NF-kappaB assay also showed a delay in the onset of capillary reactions of the allantois membrane in a physiological model for anti-inflammatory activity - the HET-CAM assay.

Various glucocorticoids differ in their ability to induce gene expression, apoptosis and to repress NF-kappaB-dependent transcription.

Glucocorticoids (GCs) influence a great variety of cellular functions by at least three important modes of action: the activation (or repression) of genes controlled by binding sites for the glucocorticoid receptor (GR), the induction of apoptosis in lymphocytes and the recently discovered cross-talk to other transcription factors such as NF-kappaB. In this study we systematically compared various natural and synthetic steroid hormones frequently used as therapeutic agents on their ability to mediate these three modes of action. Betamethasone, triamcinolone, dexamethasone and clobetasol turned out to be the best inducers of gene expression and apoptosis. All GCs including the antagonistic compound RU486 efficiently reduced NF-kappaB-mediated transactivation to comparable extents, suggesting that ligand-induced nuclear localization of the GR is sufficient for transrepression. Glucocorticoid treatment of cells did not result in elevated IkappaB-alpha expression, but impaired the tumor necrosis factor (TNF)-alpha-induced degradation of IkappaB-alpha without affecting DNA binding of NF-kappaB. The structural requirements for the various functions of glucocorticoids are discussed.

Prohibitin and prohibitone are contained in high-molecular weight complexes and interact with alpha-actinin and annexin A2.

The closely related proteins prohibitin (p32) and prohibitone (p37) are evolutionarily conserved with homologues found from cyanobacteria to man. They are thought to be exclusively mitochondrial and have been assigned many-rather different-functions, ranging from a role in lifespan, in mitochondrial inheritance and as chaperones of mitochondrial proteases in yeast. Evidence for a localisation outside of mitochondria has been brought forward in mammalian cells, where they influence cell-cycle progression and are found in association with cell surface receptors. We have employed a yeast two-hybrid screen to identify other interacting proteins and have identified alpha-actinin and annexin A2 as binding partners for prohibitin and prohibitone. Coprecipitation experiments supported the putative binding between prohibitin and prohibitone on the one hand and annexin A2 or alpha-actinin on the other hand in intact cells. Surface plasmon resonance analysis was used to determine relative affinities between prohibitin and alpha-actinin and between prohibitone and annexin A2 and alpha-actinin, respectively. We further show that prohibitin and prohibitone can also form homomeric (preferentially tetrameric) and heteromultimeric complexes, with significant affinities.

Human homeodomain-interacting protein kinase-2 (HIPK2) is a member of the DYRK family of protein kinases and maps to chromosome 7q32-q34.

Here we identified the human serine/threonine kinase HIPK2 as a novel member of the DYRK kinase subfamily. Alignment of several DYRK family proteins including the kinases minibrain, MJAK, PKY, the Dictyostelium kinase YakA and Saccharomyces YAK1 allowed the identification of several evolutionary conserved DYRK consensus motifs within the kinase domain. A lysine residue conserved between all DYRK kinase family members was found to be essential for the kinase function of HIPK2. Human HIPK2 was mapped to chromosome 7q32-q34 and murine HIPK2 to chromosome 6B, the homologue to human chromosome 7.