Trivalent and quadrivalent seasonal influenza vaccine in adults aged 60 and older: a systematic review and network meta-analysis.

OBJECTIVES: To compare the efficacy of influenza vaccines of any valency for adults 60 years and older. DESIGN AND SETTING: Systematic review with network meta-analysis (NMA) of randomised controlled trials (RCTs). MEDLINE, EMBASE, JBI Evidence-Based Practice (EBP) Database, PsycINFO, and Cochrane Evidence -Based Medicine database were searched from inception to 20 June 20, 2022. Two reviewers screened, abstracted, and appraised articles (Cochrane Risk of Bias (ROB) 2.0 tool) independently. We assessed certainty of findings using Confidence in Network Meta-Analysis and Grading of Recommendations, Assessment, Development and Evaluations approaches. We performed random-effects meta-analysis and network meta-analysis (NMA), and estimated odds ratios (ORs) for dichotomous outcomes and incidence rate ratios (IRRs) for count outcomes along with their corresponding 95% confidence intervals (CIs) and prediction intervals. PARTICIPANTS: Older adults (≥60 years old) receiving an influenza vaccine licensed in Canada or the USA (vs placebo, no vaccine, or any other licensed vaccine), at any dose. MAIN OUTCOME MEASURES: Laboratory-confirmed influenza (LCI) and influenza-like illness (ILI). Secondary outcomes were the number of vascular adverse events, hospitalisation for acute respiratory infection (ARI) and ILI, inpatient hospitalisation, emergency room (ER) visit for ILI, outpatient visit, and mortality, among others. RESULTS: We included 41 RCTs and 15 companion reports comprising 8 vaccine types and 206 032 participants. Vaccines may prevent LCI compared with placebo, with high-dose trivalent inactivated influenza vaccine (IIV3-HD) (NMA: 9 RCTs, 52 202 participants, OR 0.23, 95% confidence interval (CI) (0.11 to 0.51), low certainty of evidence) and recombinant influenza vaccine (RIV) (OR 0.25, 95%CI (0.08 to 0.73), low certainty of evidence) among the most efficacious vaccines. Standard dose trivalent IIV3 (IIV3-SD) may prevent ILI compared with placebo, but the result was imprecise (meta-analysis: 2 RCTs, 854 participants, OR 0.39, 95%CI (0.15 to 1.02), low certainty of evidence). Any HD was associated with prevention of ILI compared with placebo (NMA: 9 RCTs, 65 658 participants, OR 0.38, 95%CI (0.15 to 0.93)). Adjuvanted quadrivalent IIV (IIV4-Adj) may be associated with the least vascular adverse events, but the results were very uncertain (NMA: eight 8 RCTs, 57 677 participants, IRR 0.18, 95%CI (0.07 to 0.43), very low certainty of evidence). RIV on all-cause mortality may be comparable to placebo (NMA: 20 RCTs, 140 577 participants, OR 1.01, 95%CI (0.23 to 4.49), low certainty of evidence). CONCLUSIONS: This systematic review demonstrated efficacy associated with IIV3-HD and RIV vaccines in protecting older persons against LCI. RIV vaccine may reduce all-cause mortality when compared with other vaccines, but the evidence is uncertain. Differences in efficacy between influenza vaccines remain uncertain with very low to moderate certainty of evidence. PROSPERO REGISTRATION NUMBER: CRD42020177357.

Human sialoglycan ligands for immune inhibitory Siglecs.

Most human Siglecs (sialic acid binding immunoglobulin-like lectins) are expressed on the surfaces of overlapping subsets of immune cells, and most carry immunoreceptor tyrosine-based inhibitory domains on their intracellular motifs. When immune inhibitory Siglecs bind to complementary sialoglycans in their local milieu, engagement results in down-regulation of the immune response. Siglecs have come under scrutiny as potential targets of drugs to modify the course of inflammation (and other immune system responses) and as immune checkpoints in cancer. Human Siglecs bind to endogenous human sialoglycans. The identities of these endogenous human sialoglycan immune regulators are beginning to emerge, along with some general principles that may inform future investigations in this area. Among these principles is the finding that a cell type or tissue may express a ligand for a particular Siglec on a single or a very few of its sialoglycoproteins. The selected protein carrier for a particular Siglec may be unique in a certain tissue, but vary tissue-to-tissue. The binding affinity of endogenous Siglec ligands may surpass that of its binding to synthetic sialoglycan determinants by several orders of magnitude. Since most human Siglecs have evolved rapidly and are distinct from those in most other mammals, this review describes endogenous human Siglec ligands for several human immune inhibitory Siglecs. As the identities of these immune regulatory sialoglycan ligands are defined, additional opportunities to target Siglecs therapeutically may emerge.

Amyloid-beta and tau pathologies act synergistically to induce novel disease stage-specific microglia subtypes.

BACKGROUND: Amongst risk alleles associated with late-onset Alzheimer's disease (AD), those that converged on the regulation of microglia activity have emerged as central to disease progression. Yet, how canonical amyloid-ß (Aß) and tau pathologies regulate microglia subtypes during the progression of AD remains poorly understood. METHODS: We use single-cell RNA-sequencing to profile microglia subtypes from mice exhibiting both Aß and tau pathologies across disease progression. We identify novel microglia subtypes that are induced in response to both Aß and tau pathologies in a disease-stage-specific manner. To validate the observation in AD mouse models, we also generated a snRNA-Seq dataset from the human superior frontal gyrus (SFG) and entorhinal cortex (ERC) at different Braak stages. RESULTS: We show that during early-stage disease, interferon signaling induces a subtype of microglia termed Early-stage AD-Associated Microglia (EADAM) in response to both Aß and tau pathologies. During late-stage disease, a second microglia subtype termed Late-stage AD-Associated Microglia (LADAM) is detected. While similar microglia subtypes are observed in other models of neurodegenerative disease, the magnitude and composition of gene signatures found in EADAM and LADAM are distinct, suggesting the necessity of both Aß and tau pathologies to elicit their emergence. Importantly, the pattern of EADAM- and LADAM-associated gene expression is observed in microglia from AD brains, during the early (Braak II)- or late (Braak VI/V)- stage of the disease, respectively. Furthermore, we show that several Siglec genes are selectively expressed in either EADAM or LADAM. Siglecg is expressed in white-matter-associated LADAM, and expression of Siglec-10, the human orthologue of Siglecg, is progressively elevated in an AD-stage-dependent manner but not shown in non-AD tauopathy. CONCLUSIONS: Using scRNA-Seq in mouse models bearing amyloid-ß and/or tau pathologies, we identify novel microglia subtypes induced by the combination of Aß and tau pathologies in a disease stage-specific manner. Our findings suggest that both Aß and tau pathologies are required for the disease stage-specific induction of EADAM and LADAM. In addition, we revealed Siglecs as biomarkers of AD progression and potential therapeutic targets.

Human brain sialoglycan ligand for CD33, a microglial inhibitory Siglec implicated in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by accumulation of misfolded proteins. Genetic studies implicate microglia, brain-resident phagocytic immune cells, in AD pathogenesis. As positive effectors, microglia clear toxic proteins, whereas as negative effectors, they release proinflammatory mediators. An imbalance of these functions contributes to AD progression. Polymorphisms of human CD33, an inhibitory microglial receptor, are linked to AD susceptibility; higher CD33 expression correlates with increased AD risk. CD33, also called Siglec-3, is a member of the sialic acid-binding immunoglobulin-type lectin (Siglec) family of immune regulatory receptors. Siglec-mediated inhibition is initiated by binding to complementary sialoglycan ligands in the tissue environment. Here, we identify a single sialoglycoprotein in human cerebral cortex that binds CD33 as well as Siglec-8, the most abundant Siglec on human microglia. The ligand, which we term receptor protein tyrosine phosphatase zeta (RPTPζ)S3L, is composed of sialylated keratan sulfate chains carried on a minor isoform/glycoform of RPTPζ (phosphacan) and is found in the extracellular milieu of the human brain parenchyma. Brains from human AD donors had twofold higher levels of RPTPζS3L than age-matched control donors, raising the possibility that RPTPζS3L overexpression limits misfolded protein clearance contributing to AD pathology. Mice express the same structure, a sialylated keratan sulfate RPTPζ isoform, that binds mouse Siglec-F and crossreacts with human CD33 and Siglec-8. Brains from mice engineered to lack RPTPζ, the sialyltransferase St3gal4, or the keratan sulfate sulfotransferase Chst1 lacked Siglec binding, establishing the ligand structure. The unique CD33 and Siglec-8 ligand, RPTPζS3L, may contribute to AD progression.

Summary of the National Advisory Committee on Immunization (NACI) Supplemental Statement on Recombinant Influenza Vaccines.

Background: Recombinant protein technology is a novel platform for influenza vaccine manufacturing that differs significantly from existing egg-based and mammalian cell culture-based technologies. Supemtek™ is the first and, to date, the only recombinant quadrivalent influenza vaccine (RIV4) authorized for use in Canada in adults aged 18 years and older. The objective is to review the available evidence for efficacy, effectiveness, immunogenicity and safety of RIV4, and to summarize the National Advisory Committee on Immunization (NACI) recommendation regarding the use of Supemtek. Methods: A systematic literature review and meta-analysis on the vaccine efficacy, effectiveness, immunogenicity and safety of RIV4 in adults was conducted according to methodology specified a priori in a written protocol. NACI evidence-based process was used to assess the available evidence and develop a recommendation regarding the use of Supemtek. Results: Ten eligible studies were included in the evidence synthesis. One randomized controlled trial (RCT) in adults aged 50 years and older provided evidence that RIV4 may potentially offer improved protection against laboratory-confirmed influenza A infection compared to standard egg-based influenza vaccines. Data from eight RCTs assessing immunogenicity and five RCTs and one post-marketing surveillance study assessing safety indicated that Supemtek is a safe, well tolerated, and immunogenic alternative to conventional egg-based influenza vaccines for adults. Conclusion: There is fair evidence that Supemtek is effective, safe, and has non-inferior immunogenicity to comparable vaccines, based on direct evidence in adults 18 years of age and older; thus, NACI recommends that Supemtek may be considered among the seasonal influenza vaccines offered to adults 18 years of age and older for their annual influenza vaccination.

Siglec Ligands.

A dense and diverse array of glycans on glycoproteins and glycolipids decorate all cell surfaces. In vertebrates, many of these carry sialic acid, in a variety of linkages and glycan contexts, as their outermost sugar moiety. Among their functions, glycans engage complementary glycan binding proteins (lectins) to regulate cell physiology. Among the glycan binding proteins are the Siglecs, sialic acid binding immunoglobulin-like lectins. In humans, there are 14 Siglecs, most of which are expressed on overlapping subsets of immune system cells. Each Siglec engages distinct, endogenous sialylated glycans that initiate signaling programs and regulate cellular responses. Here, we explore the emerging science of Siglec ligands, including endogenous sialoglycoproteins and glycolipids and synthetic sialomimetics. Knowledge in this field promises to reveal new molecular pathways controlling cell physiology and new opportunities for therapeutic intervention.

Induction of the endogenous sialoglycan ligand for eosinophil death receptor Siglec-8 in chronic rhinosinusitis with hyperplastic nasal polyposis.

Siglec-8, an immune-inhibitory sialoglycan binding lectin (S8), is expressed on the surface of eosinophils and mast cells, which are potent mediators of allergic inflammation. When S8 engages endogenous sialoglycan ligands, eosinophils undergo apoptosis and mast cell mediator release is inhibited. In the human airway, Siglec-8 ligands (S8L) are sialylated keratan sulfate chains carried on isoforms of the protein Deleted in Malignant Brain Tumors-1 (DMBT1), an immunoregulatory protein that we recently identified as the endogenous ligand for S8, DMBT1S8. We herein report that S8L is overexpressed in chronic rhinosinusitis with nasal polyposis (CRSwNP), a prevalent eosinophilic laden airway disease. Quantification and comparison of the degree to which DMBT1 carries the S8L by immunoblot analysis and lectin blot overlay, respectively, from nasal lavage showed that the S8L/DMBT1 ratio was significantly increased in CRSwNP vs. control or CRS patients. We identified the histological sites of S8L and DMBT1 expression in fresh surgically resected human nasal polyps. Histochemistry of diseased polyps and adjacent nondiseased middle turbinate (MT) tissue from CRSwNP demonstrated colocalization of S8L and DMBT1 with highest staining in submucosal glands >> epithelium > stoma. S8L expression was specifically elevated in the submucosal glands and epithelium of polyp tissue compared to MT. We hypothesize that expression of the isoform of DMBT1 carrying the Siglec-8 binding sialoglycan, DMBT1S8, is induced in polyps of CRSwNP specifically at the site of disease, is produced in the submucosal glands of polyps and secreted into the lumen of the sinonasal cavity as a host response to mitigate eosinophil-mediated inflammation.

Isolation, identification, and characterization of the human airway ligand for the eosinophil and mast cell immunoinhibitory receptor Siglec-8.

BACKGROUND: The immunoinhibitory receptor Siglec-8 on the surface of human eosinophils and mast cells binds to sialic acid-containing ligands in the local milieu, resulting in eosinophil apoptosis, inhibition of mast cell degranulation, and suppression of inflammation. Siglec-8 ligands were found on postmortem human trachea and bronchi and on upper airways in 2 compartments, cartilage and submucosal glands, but they were surprisingly absent from the epithelium. We hypothesized that Siglec-8 ligands in submucosal glands and ducts are normally transported to the airway mucus layer, which is lost during tissue preparation. OBJECTIVE: Our aim was to identify the major Siglec-8 sialoglycan ligand on the mucus layer of human airways. METHODS: Human upper airway mucus layer proteins were recovered during presurgical nasal lavage of patients at a sinus clinic. Proteins were resolved by gel electrophoresis and blotted, and Siglec-8 ligands detected. Ligands were purified by size exclusion and affinity chromatography, identified by proteomic mass spectrometry, and validated by electrophoretic and histochemical colocalization. The affinity of Siglec-8 binding to purified human airway ligand was determined by inhibition of glycan binding. RESULTS: A Siglec-8-ligand with a molecular weight of approximately 1000 kDa was found in all patient nasal lavage samples. Purification and identification revealed deleted in malignant brain tumors 1 (DMBT1) (also known by the aliases GP340 and SALSA), a large glycoprotein with multiple O-glycosylation repeats. Immunoblotting, immunohistochemistry, and enzyme treatments confirmed that Siglec-8 ligand on the human airway mucus layer is an isoform of DMBT1 carrying O-linked sialylated keratan sulfate chains (DMBT1S8). Quantitative inhibition revealed that DMBT1S8 has picomolar affinity for Siglec-8. CONCLUSION: A distinct DMBT1 isoform, DMBT1S8, is the major high-avidity ligand for Siglec-8 on human airways.

Sialylated keratan sulfate proteoglycans are Siglec-8 ligands in human airways.

Human siglecs are a family of 14 sialic acid-binding proteins, most of which are expressed on subsets of immune cells where they regulate immune responses. Siglec-8 is expressed selectively on human allergic inflammatory cells-primarily eosinophils and mast cells-where engagement causes eosinophil apoptosis and inhibits mast cell mediator release. Evidence supports a model in which human eosinophils and mast cells bind to Siglec-8 sialoglycan ligands on inflammatory target tissues to resolve allergic inflammation and limit tissue damage. To identify Siglec-8-binding sialoglycans from human airways, proteins extracted from postmortem human trachea were resolved by size-exclusion chromatography and composite agarose-acrylamide gel electrophoresis, blotted and probed by Siglec-8-Fc blot overlay. Three size classes of Siglec-8 ligands were identified: 250 kDa, 600 kDa and 1 MDa, each of which was purified by affinity chromatography using a recombinant pentameric form of Siglec-8. Proteomic mass spectrometry identified all size classes as the proteoglycan aggrecan, a finding validated by immunoblotting. Glycan array studies demonstrated Siglec-8 binding to synthetic glycans with a terminal Neu5Acα2-3(6-sulfo)-Gal determinant, a quantitatively minor terminus on keratan sulfate (KS) chains of aggrecan. Treating human tracheal extracts with sialidase or keratanase eliminated Siglec-8 binding, indicating sialylated KS chains as Siglec-8-binding determinants. Treating human tracheal histological sections with keratanase also completely eliminated the binding of Siglec-8-Fc. Finally, Siglec-8 ligand purified from human trachea extracts induced increased apoptosis of freshly isolated human eosinophils in vitro. We conclude that sialylated KS proteoglycans are endogenous human airway ligands that bind Siglec-8 and may regulate allergic inflammation.

Long-Term Effects of Intermittent Adolescent Alcohol Exposure in Male and Female Rats.

Alcohol is a serious public health concern that has a differential impact on individuals depending upon age and sex. Patterns of alcohol consumption have recently changed: heavy episodic drinking-known as binge-drinking-has become most popular among the youth. Herein, we aimed to investigate the consequences of intermittent adolescent alcohol consumption in male and female animals. Thus, Wistar rats were given free access to ethanol (20% in drinking water) or tap water for 2-h sessions during 3 days, and for an additional 4-h session on the 4th day; every week during adolescence, from postnatal day (pnd) 28-52. During this period, animals consumed a moderate amount of alcohol despite blood ethanol concentration (BEC) did not achieve binge-drinking levels. No withdrawal signs were observed: no changes were observed regarding anxiety-like responses in the elevated plus-maze or plasma corticosterone levels (pnd 53-54). In the novel object recognition (NOR) test (pnd 63), a significant deficit in recognition memory was observed in both male and female rats. Western Blot analyses resulted in an increase in the expression of synaptophysin in the frontal cortex (FC) of male and female animals, together with a decrease in the expression of the CB2R in the same brain region. In addition, adolescent alcohol induced, exclusively among females, a decrease in several markers of dopaminergic and serotonergic neurotransmission, in which epigenetic mechanisms, i.e., histone acetylation, might be involved. Taken together, further research is still needed to specifically correlate sex-specific brain and behavioral consequences of adolescent alcohol exposure.

Nalmefene Prevents Alcohol-Induced Neuroinflammation and Alcohol Drinking Preference in Adolescent Female Mice: Role of TLR4.

BACKGROUND: We previously showed that, by activating innate immune receptors Toll-like 4 (TLR4), adolescent intermittent ethanol (EtOH) exposure causes neuroinflammation, myelin damage, and behavioral dysfunctions. Recent findings reveal that clinically used opioid antagonists naltrexone (NT) and naloxone (NX) inhibit opioid-induced TLR4 signaling and that NT, NX, and nalmefene (NF), the 6-methylene derivative of NX, are able to reduce alcohol drinking escalation. METHODS: NF (0.1 mg/kg, intraperitoneally) was injected 1 hour prior to EtOH (3 g/kg, intraperitoneally) following intermittent treatment in female (PND35) adolescent mice. Inflammatory molecules, myelin proteins, and apoptotic markers were assessed in the prefrontal cortex (PFC) and striatum/nucleus accumbens (STR/NAcc). The effect of NF on alcohol drinking preference was evaluated in both the wild-type and TLR4 knockout (KO) adolescent mice. Using astroglial cells, the inhibitory potential of NT, NX, and NF on lipopolysaccharide (LPS), or the EtOH-triggered TLR4 response, was compared. RESULTS: Our findings indicate that NF prevents the up-regulation of cytokines (IL-1ß, IL-17A, TNF-α), chemokines (MCP-1, MIP-1, KC), and pro-inflammatory mediators (iNOS, COX-2), along with myelin damage and apoptotic events, in both PFC and STR/NAcc. NF also abolishes EtOH-induced escalation of alcohol preference/consumption, but has no effect when administered to TLR4-KO mice. In vitro experiments indicate that NX and NF inhibit TLR4 activation upon LPS or EtOH stimulation. Immunofluorescence studies and lipid rafts isolation show that NF is able to prevent TLR4 translocation to lipid rafts/caveolae in astrocytes. CONCLUSIONS: These results suggest that NF prevents neuroinflammation and brain damage by blocking the TLR4 response and also support the role of central pro-inflammatory immune signaling in the modulation of alcohol consumption/addiction.

Siglec-8 and Siglec-9 binding specificities and endogenous airway ligand distributions and properties.

Siglecs are transmembrane sialoglycan binding proteins, most of which are expressed on leukocyte subsets and have inhibitory motifs that translate cell surface ligation into immune suppression. In humans, Siglec-8 on eosinophils, mast cells and basophils and Siglec-9 on neutrophils, monocytes and some T-cells, mediate immune cell death, inhibition of immune mediator release and/or enhancement of anti-inflammatory mediator release. Endogenous sialoglycan ligands in tissues, mostly uncharacterized, engage siglecs on leukocytes to inhibit inflammation. Glycan array analyses demonstrated that Siglec-8, Siglec-9 and their mouse counterparts Siglec-F and Siglec-E (respectively) have distinct glycan binding specificities, with Siglec-8 more structurally restricted. Since siglecs are involved in lung inflammation, we studied Siglec-8 and Siglec-9 ligands in human lungs and airways. Siglec-8 ligands are in tracheal submucosal glands and cartilage but not airway epithelium or connective tissues, whereas Siglec-9 ligands are broadly distributed. Mouse airways do not have Siglec-8 ligands, whereas Siglec-9 ligands are on airways of both species. Extraction of human airways and lung followed by electrophoretic resolution and siglec blotting revealed Siglec-8 ligands in extracts of human trachea and cultured tracheal gland cells, but not parenchyma or cultured airway epithelial cells whereas Siglec-9 ligands were extracted from all airway and lung tissues and cells tested. Siglec-8 and Siglec-9 ligands in airways appear to be high molecular weight O-linked sialoglycoproteins. These data reveal differential glycan specificities of Siglec-8, Siglec-9 and their mouse counterparts Siglec-F and Siglec-E, and the tissue distributions and molecular characteristics of Siglec-8 and Siglec-9 sialoglycan ligands on human airways and lungs.

Assessing PTEN Subcellular Localization.

PTEN subcellular localization is fundamental in the execution of the distinct PTEN biological activities, including not only its PI(3,4,5)P3 phosphatase activity when associated to membranes but also its subcellular compartment-specific interactions with regulatory and effector proteins, including those exerted in the nucleus. As a consequence, PTEN subcellular localization is tightly regulated in vivo by both intrinsic and extrinsic mechanisms. The plasma membrane/nucleus/cytoplasm partitioning of PTEN has been the focus of several studies, both from a mechanistic and from a disease-association point of view. Here, we summarize the current knowledge on PTEN plasma membrane/nucleus/cytoplasm distribution, and present subcellular fractionation, immunofluorescence, and immunohistochemical methods to study the distribution and shuttling of PTEN between these subcellular compartments.

EGF-Induced Acetylation of Heterogeneous Nuclear Ribonucleoproteins Is Dependent on KRAS Mutational Status in Colorectal Cancer Cells.

KRAS mutational status is considered a negative predictive marker of the response to anti-EGFR therapies in colorectal cancer (CRC) patients. However, conflicting data exist regarding the variable response to EGFR-targeted therapy. The effects of oncogenic KRAS on downstream targets were studied in cell lines with different KRAS mutations. Cells harboring a single KRASG13D allele showed the most tumorigenic profile, with constitutive activation of the downstream pathway, rendering them EGF-unresponsive. Conversely, KRASA146T cells showed a full EGF-response in terms of signal transduction pathways, cell proliferation, migration or adhesion. Moreover, the global acetylome of CRC cells was also dependent on KRAS mutational status. Several hnRNP family members were identified within the 36 acetylated-proteins. Acetylation status is known to be involved in the modulation of EGF-response. In agreement with results presented herein, hnRNPA1 and L acetylation was induced in response to EGF in KRASA146T cells, whereas acetyl-hnRNPA1 and L levels remained unchanged after growth factor treatment in KRASG13D unresponsive cells. Our results showed that hnRNPs induced-acetylation is dependent on KRAS mutational status. Nevertheless hnRNPs acetylation might also be the point where different oncogenic pathways converge.

A functional dissection of PTEN N-terminus: implications in PTEN subcellular targeting and tumor suppressor activity.

Spatial regulation of the tumor suppressor PTEN is exerted through alternative plasma membrane, cytoplasmic, and nuclear subcellular locations. The N-terminal region of PTEN is important for the control of PTEN subcellular localization and function. It contains both an active nuclear localization signal (NLS) and an overlapping PIP2-binding motif (PBM) involved in plasma membrane targeting. We report a comprehensive mutational and functional analysis of the PTEN N-terminus, including a panel of tumor-related mutations at this region. Nuclear/cytoplasmic partitioning in mammalian cells and PIP3 phosphatase assays in reconstituted S. cerevisiae defined categories of PTEN N-terminal mutations with distinct PIP3 phosphatase and nuclear accumulation properties. Noticeably, most tumor-related mutations that lost PIP3 phosphatase activity also displayed impaired nuclear localization. Cell proliferation and soft-agar colony formation analysis in mammalian cells of mutations with distinctive nuclear accumulation and catalytic activity patterns suggested a contribution of both properties to PTEN tumor suppressor activity. Our functional dissection of the PTEN N-terminus provides the basis for a systematic analysis of tumor-related and experimentally engineered PTEN mutations.

Expression of ligands for Siglec-8 and Siglec-9 in human airways and airway cells.

BACKGROUND: Balanced activation and inhibition of the immune system ensures pathogen clearance while avoiding hyperinflammation. Siglecs, sialic acid-binding proteins found on subsets of immune cells, often inhibit inflammation: Siglec-8 on eosinophils and Siglec-9 on neutrophils engage sialoglycan ligands on airways to diminish ongoing inflammation. The identities of human siglec ligands and their expression during inflammation are largely unknown. OBJECTIVE: The histologic distribution, expression, and molecular characteristics of siglec ligands were explored in healthy and inflamed human upper airways and in a cellular model of airway inflammation. METHODS: Normal and chronically inflamed upper airway tissues were stained for siglec ligands. The ligands were extracted from normal and inflamed tissues and from human Calu-3 cells for quantitative analysis by means of siglec blotting and isolation by means of siglec capture. RESULTS: Siglec-8 ligands were expressed on a subpopulation of submucosal gland cells of human inferior turbinate, whereas Siglec-9 ligands were expressed more broadly (submucosal glands, epithelium, and connective tissue); both were significantly upregulated in patients with chronic rhinosinusitis. Human airway (Calu-3) cells expressed Siglec-9 ligands on mucin 5B (MUC5B) under inflammatory control through the nuclear factor κB pathway, and MUC5B carried sialoglycan ligands of Siglec-9 on human upper airway tissue. CONCLUSION: Inflammation results in upregulation of immune-inhibitory Siglec-8 and Siglec-9 sialoglycan ligands on human airways. Siglec-9 ligands are upregulated through the nuclear factor κB pathway, resulting in their enhanced expression on MUC5B. Siglec sialoglycan ligand expression in inflamed cells and tissues may contribute to the control of airway inflammation.

Disulfide stress: a novel type of oxidative stress in acute pancreatitis.

Glutathione oxidation and protein glutathionylation are considered hallmarks of oxidative stress in cells because they reflect thiol redox status in proteins. Our aims were to analyze the redox status of thiols and to identify mixed disulfides and targets of redox signaling in pancreas in experimental acute pancreatitis as a model of acute inflammation associated with glutathione depletion. Glutathione depletion in pancreas in acute pancreatitis is not associated with any increase in oxidized glutathione levels or protein glutathionylation. Cystine and homocystine levels as well as protein cysteinylation and γ-glutamyl cysteinylation markedly rose in pancreas after induction of pancreatitis. Protein cysteinylation was undetectable in pancreas under basal conditions. Targets of disulfide stress were identified by Western blotting, diagonal electrophoresis, and proteomic methods. Cysteinylated albumin was detected. Redox-sensitive PP2A and tyrosine protein phosphatase activities diminished in pancreatitis and this loss was abrogated by N-acetylcysteine. According to our findings, disulfide stress may be considered a specific type of oxidative stress in acute inflammation associated with protein cysteinylation and γ-glutamylcysteinylation and oxidation of the pair cysteine/cystine, but without glutathione oxidation or changes in protein glutathionylation. Two types of targets of disulfide stress were identified: redox buffers, such as ribonuclease inhibitor or albumin, and redox-signaling thiols, which include thioredoxin 1, APE1/Ref1, Keap1, tyrosine and serine/threonine phosphatases, and protein disulfide isomerase. These targets exhibit great relevance in DNA repair, cell proliferation, apoptosis, endoplasmic reticulum stress, and inflammatory response. Disulfide stress would be a specific mechanism of redox signaling independent of glutathione redox status involved in inflammation.

PTEN increases autophagy and inhibits the ubiquitin-proteasome pathway in glioma cells independently of its lipid phosphatase activity.

Two major mechanisms of intracellular protein degradation, autophagy and the ubiquitin-proteasome pathway, operate in mammalian cells. PTEN, which is frequently mutated in glioblastomas, is a tumor suppressor gene that encodes a dual specificity phosphatase that antagonizes the phosphatidylinositol 3-kinase class I/AKT/mTOR pathway, which is a key regulator of autophagy. Here, we investigated in U87MG human glioma cells the role of PTEN in the regulation of autophagy and the ubiquitin-proteasome pathway, because both are functionally linked and are relevant in cancer progression. Since U87MG glioma cells lack a functional PTEN, we used stable clones that express, under the control of a tetracycline-inducible system (Tet-on), wild-type PTEN and two of its mutants, G129E-PTEN and C124S-PTEN, which, respectively, lack the lipid phosphatase activity only and both the lipid and the protein phosphatase activities of this protein. Expression of PTEN in U87MG glioma cells decreased proteasome activity and also reduced protein ubiquitination. On the contrary, expression of PTEN increased the autophagic flux and the lysosomal mass. Interestingly, and although PTEN negatively regulates the phosphatidylinositol 3-kinase class I/AKT/mTOR signaling pathway by its lipid phosphatase activity, both effects in U87MG cells were independent of this activity. These results suggest a new mTOR-independent signaling pathway by which PTEN can regulate in opposite directions the main mechanisms of intracellular protein degradation.

A functional network of the tumor suppressors APC, hDlg, and PTEN, that relies on recognition of specific PDZ-domains.

APC and PTEN are tumor suppressor proteins that bind through their C-termini to the PDZ domain containing-hDlg scaffolding protein. We have found that co-expression of PTEN and hDlg enhanced the negative regulation of the PI3K/Akt pathway by PTEN, indicating the physiologic importance of these interactions. APC and PTEN share other PDZ domain containing-interacting partners, including the MAGI scaffolding proteins and the MAST family of protein kinases. Mutational analysis revealed that the C-terminal PDZ-binding motifs from APC and PTEN were differentially recognized by distinct PDZ domains. APC bound to the three PDZ domains from hDlg, whereas PTEN mainly bound to PDZ-2/hDlg. This indicates the existence of overlapping, but distinct PDZ-domain recognition patterns by APC and PTEN. Furthermore, a ternary complex formed by APC, PTEN, and hDlg was detected, suggesting that hDlg may serve as a platform to bring in proximity APC and PTEN tumor suppressor activities. In line with this, tumor-related mutations targeting the PDZ-2/hDlg domain diminished its interaction with APC and PTEN. Our results expand the PDZ-domain counterparts for the tumor suppressor APC, show that APC and PTEN share PDZ-domain partners but have individual molecular determinants for specific recognition of PDZ domains, and suggest the participation of the tumor suppressors APC, PTEN, and hDlg in PDZ-domain interaction networks which may be relevant in oncogenesis.

Cytoplasmic p27Kip1 counteracts the pro-apoptotic function of the open conformation of PTEN by retention and destabilization of PTEN outside of the nucleus.

The tumor suppressor activity of p27Kip1 takes place in the cell nucleus by inhibitory binding to cyclin/CDK complexes. p27Kip1 can also be localized in the cytoplasm, where it has been proposed to have oncogenic properties. Here, we describe a novel role for cytoplasmic p27Kip1 which could account for its activity as an oncoprotein by negative regulation of the PTEN tumor suppressor. p27Kip1 physically interacted with the open conformation of PTEN, which is competent to enter the nucleus. In mammalian cells, cytoplasmic p27Kip1 retained to nuclear-targeted PTEN in the cytoplasm. This retention was exerted by the C-terminal p27Kip1 region, and was independent of cyclin/CDK-binding. The nuclear accumulation of PTEN triggered by pro-apoptotic TNFα treatment was abolished by cytoplasmic p27Kip1. Furthermore, conformationally-open PTEN displayed diminished protein stability and pro-apoptotic activity in the presence of cytoplasmic p27Kip1. Our results support a conformationally-dependent model of cytoplasmic retention and negative regulation of the activity of nuclear PTEN by oncogenic cytoplasmic p27Kip1, and suggest the existence of reciprocal mechanisms to regulate the levels of both p27Kip1 and PTEN.