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.

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.

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.

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.

Deregulated expression of TANK in glioblastomas triggers pro-tumorigenic ERK1/2 and AKT signaling pathways.

Signal transmission by the noncanonical IkappaB kinases (IKKs), TANK-binding kinase 1 (TBK1) and IKKɛ, requires interaction with adapter proteins such as TRAF associated NF-κB activator (TANK). Although increased expression or dysregulation of both kinases has been described for a variety of human cancers, this study shows that deregulated expression of the TANK protein is frequently occurring in glioblastomas (GBMs). The functional relevance of TANK was analyzed in a panel of GBM-derived cell lines and revealed that knockdown of TANK arrests cells in the S-phase and prohibits tumor cell migration. Deregulated TANK expression affects several signaling pathways controlling cell proliferation and the inflammatory response. Interference with stoichiometrically assembled signaling complexes by overexpression or silencing of TANK prevented constitutive interferon-regulatory factor 3 (IRF3) phosphorylation. Knockdown of TANK frequently prevents constitutive activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). TANK-mediated ERK1/2 activation is independent from the canonical MAP kinase or ERK kinase (MEK) 1/2-mediated pathway and utilizes an alternative pathway that uses a TBK1/IKKɛ/Akt signaling axis, thus identifying a novel pathway suitable to block constitutive ERK1/2 activity.

Regulation of NF-κB activity by competition between RelA acetylation and ubiquitination.

The nuclear factor (NF)-κB transcription factor has essential roles in inflammation and oncogenesis. Its ubiquitous RelA subunit is regulated by several post-translational modifications, including phosphorylation, ubiquitination and acetylation. Ubiquitination promotes the termination of RelA-dependent transcription, but its regulation is incompletely understood. Through mass spectrometry analysis of ubiquitinated RelA, we identified seven lysines that were attached to degradative and non-degradative forms of polyubiquitin. Interestingly, lysines targeted for acetylation were among the residues identified as ubiquitin acceptor sites. Mutation of these particular sites resulted in decreased polyubiquitination. Acetylation and ubiquitination were found to inhibit each other, consistent with their use of overlapping sites. Reconstitution of rela(-/-) fibroblasts with wild-type and mutant forms of RelA revealed that modifications at these residues can have activating and inhibitory functions depending on the target gene context. Altogether, this study elucidates that ubiquitination and acetylation can modulate each other and regulate nuclear NF-κB function in a gene-specific manner.

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.

The human protein kinase HIPK2 phosphorylates and downregulates the methyl-binding transcription factor ZBTB4.

HIPK2 is a eukaryotic Serine-Threonine kinase that controls cellular proliferation and survival in response to exogenous signals. Here, we show that the human transcription factor ZBTB4 is a new target of HIPK2. The two proteins interact in vitro, colocalize and associate in vivo, and HIPK2 phosphorylates several conserved residues of ZBTB4. Overexpressing HIPK2 causes the degradation of ZBTB4, whereas overexpressing a kinase-deficient mutant of HIPK2 has no effect. The chemical activation of HIPK2 also decreases the amount of ZBTB4 in cells. Conversely, the inhibition of HIPK2 by drugs or by RNA interference causes a large increase in ZBTB4 levels. This negative regulation of ZBTB4 by HIPK2 occurs under normal conditions of cell growth. In addition, the degradation is increased by DNA damage. These findings have two consequences. First, we have recently shown that ZBTB4 inhibits the transcription of p21. Therefore, the activation of p21 by HIPK2 is two-pronged: stimulation of the activator p53, and simultaneous repression of the inhibitor ZBTB4. Second, ZBTB4 is also known to bind methylated DNA and repress methylated sequences. Consequently, our findings raise the possibility that HIPK2 might influence the epigenetic regulation of gene expression at loci that remain to be identified.

PML tumor suppressor is regulated by HIPK2-mediated phosphorylation in response to DNA damage.

The promyelocytic leukemia (PML) tumor suppressor protein, a central regulator of cell proliferation and apoptosis, is frequently fused to the retinoic acid receptor-alpha (RARalpha) in acute PML. Here we show the interaction of PML with another tumor suppressor protein, the serine/threonine kinase homeodomain-interacting protein kinase (HIPK2). In response to DNA damage, HIPK2 phosphorylates PML at serines 8 and 38. Although HIPK2-mediated phosphorylation of PML occurs early during the DNA damage response, the oncogenic PML-RARalpha fusion protein is phosphorylated with significantly delayed kinetics. DNA damage or HIPK2 expression leads to the stabilization of PML and PML-RARalpha proteins. The N-terminal phosphorylation sites contribute to the DNA damage-induced PML SUMOylation and are required for the ability of PML to cooperate with HIPK2 for the induction of cell death.

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.

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.

The slipping rib syndrome in children.

The slipping rib syndrome is an infrequent cause of thoracic and upper abdominal pain and is thought to arise from the inadequacy or rupture of the interchondral fibrous attachments of the anterior ribs. This disruption allows the costal cartilage tips to sublux, impinging on the intercostal nerves. Children with this entity are seldom described in the literature. We present a retrospective review of 12 children and young adults with slipping rib syndrome and a systematic approach for evaluation and treatment.

Experience with an anesthesiologist interventional model for endoscopy in a pediatric hospital.

BACKGROUND: Endoscopy is now a routine part of the work-up for many patients with gastrointestinal symptoms. Adults tolerate these procedures well, with either no sedation or a relatively light level. In contrast, children often require deep sedation or a general anesthetic to successfully perform these procedures. Therefore, pediatric endoscopies may require more time, personnel, and monitoring equipment to provide optimal conditions for the patient. The goals of this retrospective case series were to describe the anesthesia times and recovery duration of the different procedures, the types and amounts of medications commonly used, and the types and rates of complications experienced. METHODS: Patients (2,306) who underwent endoscopy in the Arkansas Children's Hospital endoscopy suite during a 4-year period were identified. A random sample of 720 charts was reviewed retrospectively. RESULTS: Patients ranged in age from younger than 1 year to 29 years. Patients most often had abdominal pain or multiple gastrointestinal symptoms. Sixty-eight percent of patients underwent esophagogastroduodenoscopies; 30% colonoscopy or a combination of the two. Ninety-five percent of patients received a propofol-based anesthetic. Midazolam, fentanyl, and alfentanil were frequently used as supplemental agents. Complications occurred infrequently and were airway related. All complications were easily treated, with no adverse sequelae. CONCLUSIONS: This model of anesthesiologist-provided sedation/anesthesia for gastrointestinal endoscopy procedures has been extremely successful in the Arkansas Children's Hospital and has served to heighten awareness of many issues surrounding sedation and anesthesia outside of the operating room, while ensuring a high level of care is provided.

CD95-induced JNK activation signals are transmitted by the death-inducing signaling complex (DISC), but not by Daxx.

Here we investigated CD95-mediated JNK activation pathways and their physiological relevance by employing a variety of cell lines with deficiencies in individual signal transmitting proteins. JNK activation was completely dependent on the activation of caspases in type I and type II cells, as revealed by the inhibitory effects of the caspase inhibitors zVAD-fmk or the cowpoxvirus-encoded CrmA protein. Jurkat cells deficient in caspase-8 or expressing a dominant negative (DN) form of FADD were unable to induce JNK in response to CD95 ligation, indicating that these death-inducing signaling complex (DISC) proteins are required for signal transmission. Activation of caspases, JNK and apoptosis occurred with a markedly slower kinetics in cells expressing a DN version of ASK1, revealing an important contribution of ASK1 for these processes. A C-terminally truncated version of Daxx impaired CD95-mediated apoptosis without affecting the JNK signal. DN forms of FADD, MKK4 and MKK7 completely inhibited CD95-mediated JNK activation but remained without impact on cell killing, indicating that JNK activation is not required for the execution process of CD95-mediated cell killing.

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.

Pheophorbide A from Solanum diflorum interferes with NF-kappa B activation.

Continuing our search for biogenic NF-kappa B inhibitors we investigated Solanum diflorum, used by the Istmo Sierra Zapotec Indians of Mexico in the treatment of inflammatory skin conditions. It became obvious very early that the active substance seems to be a degradation product of chlorophyll. Pheophorbide A was identified as one of the key compounds responsible for the NF-kappa B inhibitory activity. The compound interferes with NF-kappa B activation, was cytotoxic if exposed to light, but devoid of any cytotoxic activity in the dark.

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.

Protein kinase C theta cooperates with Vav1 to induce JNK activity in T-cells.

Here we show that in human T-cell leukemia cells Vav1 and protein kinase C theta (PKCtheta) synergize for the activation of c-Jun N-terminal kinase (JNK) but not p38 MAP kinase. Vav1 and PKCtheta also cooperated to induce transcription of reporter genes controlled either by AP-1 binding sites or the CD28RE/AP composite element contained in the IL-2 promoter by stimulating the binding of transcription factors to these two elements. Dominant negative versions of Vav1 and PKCtheta inhibited CD3/CD28-induced activation of JNK, revealing their relative importance for this activation pathway. Gel filtration experiments revealed the existence of constitutively associated Vav1/PKCtheta heterodimers in extracts from unstimulated T-cells, whereas T-cell costimulation induced the recruitment of Vav1 into high molecular weight complexes. Several experimental approaches showed that Vav1 is located upstream from PKCtheta in the control of the pathway leading to synergistic JNK activation. Vav1-derived signals lead to the activation of JNK by at least two different pathways. The major contribution of Vav1 for the activation of JNK relies on the PKCtheta-mediated Ca(2+)-independent synergistic activation pathway, whereas JNK is also activated by a separate Ca(2+)-dependent signaling route.

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.