MmuPV1 E7's interaction with PTPN14 delays Epithelial differentiation and contributes to virus-induced skin disease.

Human papillomaviruses (HPVs) contribute to approximately 5% of all human cancers. Species-specific barriers limit the ability to study HPV pathogenesis in animal models. Murine papillomavirus (MmuPV1) provides a powerful tool to study the roles of papillomavirus genes in pathogenesis arising from a natural infection. We previously identified Protein Tyrosine Phosphatase Non-Receptor Type 14 (PTPN14), a tumor suppressor targeted by HPV E7 proteins, as a putative cellular target of MmuPV1 E7. Here, we confirmed the MmuPV1 E7-PTPN14 interaction. Based on the published structure of the HPV18 E7/PTPN14 complex, we generated a MmuPV1 E7 mutant, E7K81S, that was defective for binding PTPN14. Wild-type (WT) and E7K81S mutant viral genomes replicated as extrachromosomal circular DNAs to comparable levels in mouse keratinocytes. E7K81S mutant virus (E7K81S MmuPV1) was generated and used to infect FoxN/Nude mice. E7K81S MmuPV1 caused neoplastic lesions at a frequency similar to that of WT MmuPV1, but the lesions arose later and were smaller than WT-induced lesions. The E7K81S MmuPV1-induced lesions also had a trend towards a less severe grade of neoplastic disease. In the lesions, E7K81S MmuPV1 supported the late (productive) stage of the viral life cycle and promoted E2F activity and cellular DNA synthesis in suprabasal epithelial cells to similar degrees as WT MmuPV1. There was a similar frequency of lateral spread of infections among mice infected with E7K81S or WT MmuPV1. Compared to WT MmuPV1-induced lesions, E7K81S MmuPV1-induced lesions had a significant expansion of cells expressing differentiation markers, Keratin 10 and Involucrin. We conclude that an intact PTPN14 binding site is necessary for MmuPV1 E7's ability to contribute to papillomavirus-induced pathogenesis and this correlates with MmuPV1 E7 causing a delay in epithelial differentiation, which is a hallmark of papillomavirus-induced neoplasia.

Reduced IRF4 expression promotes lytic phenotype in Type 2 EBV-infected B cells.

Humans are infected with two types of EBV (Type 1 (T1) and Type 2 (T2)) that differ substantially in their EBNA2 and EBNA 3A/B/C latency proteins and have different phenotypes in B cells. T1 EBV transforms B cells more efficiently than T2 EBV in vitro, and T2 EBV-infected B cells are more lytic. We previously showed that both increased NFATc1/c2 activity, and an NFAT-binding motif within the BZLF1 immediate-early promoter variant (Zp-V3) contained in all T2 strains, contribute to lytic infection in T2 EBV-infected B cells. Here we compare cellular and viral gene expression in early-passage lymphoblastoid cell lines (LCLs) infected with either T1 or T2 EBV strains. Using bulk RNA-seq, we show that T2 LCLs are readily distinguishable from T1 LCLs, with approximately 600 differentially expressed cellular genes. Gene Set Enrichment Analysis (GSEA) suggests that T2 LCLs have increased B-cell receptor (BCR) signaling, NFAT activation, and enhanced expression of epithelial-mesenchymal-transition-associated genes. T2 LCLs also have decreased RNA and protein expression of a cellular gene required for survival of T1 LCLs, IRF4. In addition to its essential role in plasma cell differentiation, IRF4 decreases BCR signaling. Knock-down of IRF4 in a T1 LCL (infected with the Zp-V3-containing Akata strain) induced lytic reactivation whereas over-expression of IRF4 in Burkitt lymphoma cells inhibited both NFATc1 and NFATc2 expression and lytic EBV reactivation. Single-cell RNA-seq confirmed that T2 LCLs have many more lytic cells compared to T1 LCLs and showed that lytically infected cells have both increased NFATc1, and decreased IRF4, compared to latently infected cells. These studies reveal numerous differences in cellular gene expression in B cells infected with T1 versus T2 EBV and suggest that decreased IRF4 contributes to both the latent and lytic phenotypes in cells with T2 EBV.

B cells infected with Type 2 Epstein-Barr virus (EBV) have increased NFATc1/NFATc2 activity and enhanced lytic gene expression in comparison to Type 1 EBV infection.

Humans are infected with two distinct strains (Type 1 (T1) and Type 2 (T2)) of Epstein-Barr virus (EBV) that differ substantially in their EBNA2 and EBNA 3A/B/C latency genes and the ability to transform B cells in vitro. While most T1 EBV strains contain the "prototype" form of the BZLF1 immediate-early promoter ("Zp-P"), all T2 strains contain the "Zp-V3" variant, which contains an NFAT binding motif and is activated much more strongly by B-cell receptor signalling. Whether B cells infected with T2 EBV are more lytic than cells infected with T1 EBV is unknown. Here we show that B cells infected with T2 EBV strains (AG876 and BL5) have much more lytic protein expression compared to B cells infected with T1 EBV strains (M81, Akata, and Mutu) in both a cord blood-humanized (CBH) mouse model and EBV-transformed lymphoblastoid cell lines (LCLs). Although T2 LCLs grow more slowly than T1 LCLs, both EBV types induce B-cell lymphomas in CBH mice. T1 EBV strains (M81 and Akata) containing Zp-V3 are less lytic than T2 EBV strains, suggesting that Zp-V3 is not sufficient to confer a lytic phenotype. Instead, we find that T2 LCLs express much higher levels of activated NFATc1 and NFATc2, and that cyclosporine (an NFAT inhibitor) and knockdown of NFATc2 attenuate constitutive lytic infection in T2 LCLs. Both NFATc1 and NFATc2 induce lytic EBV gene expression when combined with activated CAMKIV (which is activated by calcium signaling and activates MEF2D) in Burkitt Akata cells. Together, these results suggest that B cells infected with T2 EBV are more lytic due to increased activity of the cellular NFATc1/c2 transcription factors in addition to the universal presence of the Zp-V3 form of BZLF1 promoter.

EBNA2-deleted Epstein-Barr virus (EBV) isolate, P3HR1, causes Hodgkin-like lymphomas and diffuse large B cell lymphomas with type II and Wp-restricted latency types in humanized mice.

EBV transforms B cells in vitro and causes human B-cell lymphomas including classical Hodgkin lymphoma (CHL), Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL). The EBV latency protein, EBNA2, transcriptionally activates the promoters of all latent viral protein-coding genes expressed in type III EBV latency and is essential for EBV's ability to transform B cells in vitro. However, EBNA2 is not expressed in EBV-infected CHLs and BLs in humans. EBV-positive CHLs have type II latency and are largely driven by the EBV LMP1/LMP2A proteins, while EBV-positive BLs, which usually have type I latency are largely driven by c-Myc translocations, and only express the EBNA1 protein and viral non-coding RNAs. Approximately 15% of human BLs contain naturally occurring EBNA2-deleted viruses that support a form of viral latency known as Wp-restricted (expressing the EBNA-LP, EBNA3A/3B/3C, EBNA1 and BHRF1 proteins), but whether Wp-restricted latency and/or EBNA2-deleted EBV can induce lymphomas in humanized mice, or in the absence of c-Myc translocations, is unknown. Here we show that a naturally occurring EBNA2-deleted EBV strain (P3HR1) isolated from a human BL induces EBV-positive B-cell lymphomas in a subset of infected cord blood-humanized (CBH) mice. Furthermore, we find that P3HR1-infected lymphoma cells support two different viral latency types and phenotypes that are mutually exclusive: 1) Large (often multinucleated), CD30-positive, CD45-negative cells reminiscent of the Reed-Sternberg (RS) cells in CHL that express high levels of LMP1 but not EBNA-LP (consistent with type II viral latency); and 2) smaller monomorphic CD30-negative DLBCL-like cells that express EBNA-LP and EBNA3A but not LMP1 (consistent with Wp-restricted latency). These results reveal that EBNA2 is not absolutely required for EBV to form tumors in CBH mice and suggest that P3HR1 virus can be used to model EBV positive lymphomas with both Wp-restricted and type II latency in vivo.

Do Dual-mobility Cups Reduce Revision Risk in Femoral Neck Fractures Compared With Conventional THA Designs? An International Meta-analysis of Arthroplasty Registries.

BACKGROUND: Dual-mobility cups in THA were designed to reduce prosthesis instability and the subsequent risk of revision surgery in high-risk patients, such as those with hip fractures. However, there are limited data from clinical studies reporting a revision benefit of dual-mobility over conventional THA. Collaboration between anthroplasty registries provides an opportunity to describe international practice variation and compare between-country, all-cause revision rates for dual-mobility and conventional THA. QUESTIONS/PURPOSES: We summarized observational data from multiple arthroplasty registries for patients receiving either a dual-mobility or conventional THA to ask: (1) Is dual-mobility use associated with a difference in risk of all-cause revision surgery compared with conventional THA? (2) Are there specific patient characteristics associated with dual-mobility use in the hip fracture population? (3) Has the use of dual-mobility constructs changed over time in patients receiving a THA for hip fracture? METHODS: Six member registries of the International Society of Arthroplasty Registries (from Australia, Denmark, Sweden, the Netherlands, the United Kingdom, and the United States) provided custom aggregate data reports stratified by acetabular cup type (dual-mobility or conventional THA) in primary THA for hip fracture between January 1, 2002, and December 31, 2019; surgical approach; and patient demographic data (sex, mean age, American Society of Anesthesiologists class, and BMI). The cumulative percent revision and mortality were calculated for each registry. To determine a global hazard ratio of all-cause revision for dual-mobility compared with conventional THA designs, we used a pseudoindividual patient data approach to pool Kaplan-Meier prosthesis revision data from each registry and perform a meta-analysis. The pseudoindividual patient data approach is a validated technique for meta-analysis of aggregate time-to-event survival data, such as revision surgery, from multiple sources. Data were available for 15,024 dual-mobility THAs and 97,200 conventional THAs performed for hip fractures during the study period. RESULTS: After pooling of complete Kaplan-Meier survival data from all six registries, the cumulative percent revision for conventional THA was 4.3% (95% confidence interval [CI] 4.2% to 4.5%) and 4.7% (95% CI 4.3% to 5.3%) for dual-mobility THA at 5 years. We did not demonstrate a lower risk of all-cause revision for patients receiving dual-mobility over conventional THA designs for hip fracture in the meta-analysis once between-registry differences were adjusted for (HR 0.96 [95% CI 0.86 to 1.06]). A lower proportion of dual-mobility procedures were revised for dislocation than conventional THAs (0.9% versus 1.4%) but a higher proportion were revised for infection (1.2% versus 0.8%). In most registries, a greater proportion of dual-mobility THA patients were older, had more comorbidities, and underwent a posterior approach compared with conventional THA (p < 0.001). The proportion of dual-mobility THA used to treat hip fractures increased in each registry over time and constituted 21% (2438 of 11,874) of all THA procedures in 2019. CONCLUSION: The proportion of dual-mobility THAs in patients with hip fractures increased over time, but there was large variation in use across countries represented here. Dual-mobility cups were not associated with a reduction in the overall risk of revision surgery in patients with hip fractures. A randomized controlled trial powered to detect the incidence of dislocation and subsequent revision surgery is required to clarify the efficacy of dual-mobility cups to treat hip fractures. LEVEL OF EVIDENCE: Level III, therapeutic study.

An EBNA3A-Mutated Epstein-Barr Virus Retains the Capacity for Lymphomagenesis in a Cord Blood-Humanized Mouse Model.

Epstein-Barr virus (EBV) causes B cell lymphomas and transforms B cells in vitro The EBV protein EBNA3A collaborates with EBNA3C to repress p16 expression and is required for efficient transformation in vitro An EBNA3A deletion mutant EBV strain was recently reported to establish latency in humanized mice but not cause tumors. Here, we compare the phenotypes of an EBNA3A mutant EBV (Δ3A) and wild-type (WT) EBV in a cord blood-humanized (CBH) mouse model. The hypomorphic Δ3A mutant, in which a stop codon is inserted downstream from the first ATG and the open reading frame is disrupted by a 1-bp insertion, expresses very small amounts of EBNA3A using an alternative ATG at residue 15. Δ3A caused B cell lymphomas at rates similar to their induction by WT EBV but with delayed onset. Δ3A and WT tumors expressed equivalent levels of EBNA2 and p16, but Δ3A tumors in some cases had reduced LMP1. Like the WT EBV tumors, Δ3A lymphomas were oligoclonal/monoclonal, with typically one dominant IGHV gene being expressed. Transcriptome sequencing (RNA-seq) analysis revealed small but consistent gene expression differences involving multiple cellular genes in the WT EBV- versus Δ3A-infected tumors and increased expression of genes associated with T cells, suggesting increased T cell infiltration of tumors. Consistent with an impact of EBNA3A on immune function, we found that the expression of CLEC2D, a receptor that has previously been shown to influence responses of T and NK cells, was markedly diminished in cells infected with EBNA3A mutant virus. Together, these studies suggest that EBNA3A contributes to efficient EBV-induced lymphomagenesis in CBH mice.IMPORTANCE The EBV protein EBNA3A is expressed in latently infected B cells and is important for efficient EBV-induced transformation of B cells in vitro In this study, we used a cord blood-humanized mouse model to compare the phenotypes of an EBNA3A hypomorph mutant virus (Δ3A) and wild-type EBV. The Δ3A virus caused lymphomas with delayed onset compared to the onset of those caused by WT EBV, although the tumors occurred at a similar rate. The WT EBV and EBNA3A mutant tumors expressed similar levels of the EBV protein EBNA2 and cellular protein p16, but in some cases, Δ3A tumors had less LMP1. Our analysis suggested that Δ3A-infected tumors have elevated T cell infiltrates and decreased expression of the CLEC2D receptor, which may point to potential novel roles of EBNA3A in T cell and NK cell responses to EBV-infected tumors.

An EBNA3C-deleted Epstein-Barr virus (EBV) mutant causes B-cell lymphomas with delayed onset in a cord blood-humanized mouse model.

EBV causes human B-cell lymphomas and transforms B cells in vitro. EBNA3C, an EBV protein expressed in latently-infected cells, is required for EBV transformation of B cells in vitro. While EBNA3C undoubtedly plays a key role in allowing EBV to successfully infect B cells, many EBV+ lymphomas do not express this protein, suggesting that cellular mutations and/or signaling pathways may obviate the need for EBNA3C in vivo under certain conditions. EBNA3C collaborates with EBNA3A to repress expression of the CDKN2A-encoded tumor suppressors, p16 and p14, and EBNA3C-deleted EBV transforms B cells containing a p16 germline mutation in vitro. Here we have examined the phenotype of an EBNAC-deleted virus (Δ3C EBV) in a cord blood-humanized mouse model (CBH). We found that the Δ3C virus induced fewer lymphomas (occurring with a delayed onset) in comparison to the wild-type (WT) control virus, although a subset (10/26) of Δ3C-infected CBH mice eventually developed invasive diffuse large B cell lymphomas with type III latency. Both WT and Δ3C viruses induced B-cell lymphomas with restricted B-cell populations and heterogeneous T-cell infiltration. In comparison to WT-infected tumors, Δ3C-infected tumors had greatly increased p16 levels, and RNA-seq analysis revealed a decrease in E2F target gene expression. However, we found that Δ3C-infected tumors expressed c-Myc and cyclin E at similar levels compared to WT-infected tumors, allowing cells to at least partially bypass p16-mediated cell cycle inhibition. The anti-apoptotic proteins, BCL2 and IRF4, were expressed in Δ3C-infected tumors, likely helping cells avoid c-Myc-induced apoptosis. Unexpectedly, Δ3C-infected tumors had increased T-cell infiltration, increased expression of T-cell chemokines (CCL5, CCL20 and CCL22) and enhanced type I interferon response in comparison to WT tumors. Together, these results reveal that EBNA3C contributes to, but is not essential for, EBV-induced lymphomagenesis in CBH mice, and suggest potentially important immunologic roles of EBNA3C in vivo.

A cancer-associated Epstein-Barr virus BZLF1 promoter variant enhances lytic infection.

Latent Epstein-Barr virus (EBV) infection contributes to both B-cell and epithelial-cell malignancies. However, whether lytic EBV infection also contributes to tumors is unclear, although the association between malaria infection and Burkitt lymphomas (BLs) may involve excessive lytic EBV replication. A particular variant of the viral promoter (Zp) that controls lytic EBV reactivation is over-represented, relative to its frequency in non-malignant tissue, in EBV-positive nasopharyngeal carcinomas and AIDS-related lymphomas. To date, no functional differences between the prototype Zp (Zp-P) and the cancer-associated variant (Zp-V3) have been identified. Here we show that a single nucleotide difference between the Zp-V3 and Zp-P promoters creates a binding site for the cellular transcription factor, NFATc1, in the Zp-V3 (but not Zp-P) variant, and greatly enhances Zp activity and lytic viral reactivation in response to NFATc1-inducing stimuli such as B-cell receptor activation and ionomycin. Furthermore, we demonstrate that restoring this NFATc1-motif to the Zp-P variant in the context of the intact EBV B95.8 strain genome greatly enhances lytic viral reactivation in response to the NFATc1-activating agent, ionomycin, and this effect is blocked by the NFAT inhibitory agent, cyclosporine, as well as NFATc1 siRNA. We also show that the Zp-V3 variant is over-represented in EBV-positive BLs and gastric cancers, and in EBV-transformed B-cell lines derived from EBV-infected breast milk of Kenyan mothers that had malaria during pregnancy. These results demonstrate that the Zp-V3 enhances EBV lytic reactivation to physiologically-relevant stimuli, and suggest that increased lytic infection may contribute to the increased prevalence of this variant in EBV-associated malignancies.

Hypoxia-inducible factor-1α plays roles in Epstein-Barr virus's natural life cycle and tumorigenesis by inducing lytic infection through direct binding to the immediate-early BZLF1 gene promoter.

When confronted with poor oxygenation, cells adapt by activating survival signaling pathways, including the oxygen-sensitive transcriptional regulators called hypoxia-inducible factor alphas (HIF-αs). We report here that HIF-1α also regulates the life cycle of Epstein-Barr virus (EBV). Incubation of EBV-positive gastric carcinoma AGS-Akata and SNU-719 and Burkitt lymphoma Sal and KemIII cell lines with a prolyl hydroxylase inhibitor, L-mimosine or deferoxamine, or the NEDDylation inhibitor MLN4924 promoted rapid and sustained accumulation of both HIF-1α and lytic EBV antigens. ShRNA knockdown of HIF-1α significantly reduced deferoxamine-mediated lytic reactivation. HIF-1α directly bound the promoter of the EBV primary latent-lytic switch BZLF1 gene, Zp, activating transcription via a consensus hypoxia-response element (HRE) located at nt -83 through -76 relative to the transcription initiation site. HIF-1α did not activate transcription from the other EBV immediate-early gene, BRLF1. Importantly, expression of HIF-1α induced EBV lytic-gene expression in cells harboring wild-type EBV, but not in cells infected with variants containing base-pair substitution mutations within this HRE. Human oral keratinocyte (NOK) and gingival epithelial (hGET) cells induced to differentiate by incubation with either methyl cellulose or growth in organotypic culture accumulated both HIF-1α and Blimp-1α, another cellular factor implicated in lytic reactivation. HIF-1α activity also accumulated along with Blimp-1α during B-cell differentiation into plasma cells. Furthermore, most BZLF1-expressing cells observed in lymphomas induced by EBV in NSG mice with a humanized immune system were located distal to blood vessels in hypoxic regions of the tumors. Thus, we conclude that HIF-1α plays central roles in both EBV's natural life cycle and EBV-associated tumorigenesis. We propose that drugs that induce HIF-1α protein accumulation are good candidates for development of a lytic-induction therapy for treating some EBV-associated malignancies.

Coding algorithms for defining Charlson and Elixhauser co-morbidities in Read-coded databases.

BACKGROUND: Comorbidity measures, such as the Charlson Comorbidity Index (CCI) and Elixhauser Method (EM), are frequently used for risk-adjustment by healthcare researchers. This study sought to create CCI and EM lists of Read codes, which are standard terminology used in some large primary care databases. It also aimed to describe and compare the predictive properties of the CCI and EM amongst patients with hip fracture (and matched controls) in a large primary care administrative dataset. METHODS: Two researchers independently screened 111,929 individual Read codes to populate the 17 CCI and 31 EM comorbidity categories. Patients with hip fractures were identified (together with age- and sex-matched controls) from UK primary care practices participating in the Clinical Practice Research Datalink (CPRD). The predictive properties of both comorbidity measures were explored in hip fracture and control populations using logistic regression models fitted with 30- and 365-day mortality as the dependent variables together with tests of equality for Receiver Operating Characteristic (ROC) curves. RESULTS: There were 5832 CCI and 7156 EM comorbidity codes. The EM improved the ability of a logistic regression model (using age and sex as covariables) to predict 30-day mortality (AUROC 0.744 versus 0.686). The EM alone also outperformed the CCI (0.696 versus 0.601). Capturing comorbidities over a prolonged period only modestly improved the predictive value of either index: EM 1-year look-back 0.645 versus 5-year 0.676 versus complete record 0.695 and CCI 0.574 versus 0.591 versus 0.605. CONCLUSIONS: The comorbidity code lists may be used by future researchers to calculate CCI and EM using records from Read coded databases. The EM is preferable to the CCI but only marginal gains should be expected from incorporating comorbidities over a period longer than 1 year.

Development of a novel inducer for EBV lytic therapy.

Epstein-Barr virus (EBV) is a human herpesvirus that infects over 90% of the world's population that persists as a latent infection in various lymphoid and epithelial malignancies. The total number of EBV associated malignancies is estimated to exceed 200,000 new cancers per year. Current chemotherapeutic treatments of EBV-positive cancers include broad-spectrum cytotoxic drugs that ignore the EBV positive status of tumors and have limited safety and selectivity. In an effort to develop new and more efficacious molecules for inducing EBV reactivation, we have developed high-throughput screening assays to identify a class of small molecules (referred to as the C60 series) that efficiently activate the EBV lytic cycle in multiple latency types, including lymphoblastoid and nasopharyngeal carcinoma cell lines. In this paper we report our preliminary structure activity relationship studies and demonstrate reactivation of EBV in the SNU719 gastric carcinoma mouse model and the AGS-Akata gastric carcinoma mouse model.

Latent Membrane Protein 1 (LMP1) and LMP2A Collaborate To Promote Epstein-Barr Virus-Induced B Cell Lymphomas in a Cord Blood-Humanized Mouse Model but Are Not Essential.

Epstein-Barr virus (EBV) infection is associated with B cell lymphomas in humans. The ability of EBV to convert human B cells into long-lived lymphoblastoid cell lines (LCLs) in vitro requires the collaborative effects of EBNA2 (which hijacks Notch signaling), latent membrane protein 1 (LMP1) (which mimics CD40 signaling), and EBV-encoded nuclear antigen 3A (EBNA3A) and EBNA3C (which inhibit oncogene-induced senescence and apoptosis). However, we recently showed that an LMP1-deleted EBV mutant induces B cell lymphomas in a newly developed cord blood-humanized mouse model that allows EBV-infected B cells to interact with CD4 T cells (the major source of CD40 ligand). Here we examined whether the EBV LMP2A protein, which mimics constitutively active B cell receptor signaling, is required for EBV-induced lymphomas in this model. We find that the deletion of LMP2A delays the onset of EBV-induced lymphomas but does not affect the tumor phenotype or the number of tumors. The simultaneous deletion of both LMP1 and LMP2A results in fewer tumors and a further delay in tumor onset. Nevertheless, the LMP1/LMP2A double mutant induces lymphomas in approximately half of the infected animals. These results indicate that neither LMP1 nor LMP2A is absolutely essential for the ability of EBV to induce B cell lymphomas in the cord blood-humanized mouse model, although the simultaneous loss of both LMP1 and LMP2A decreases the proportion of animals developing tumors and increases the time to tumor onset. Thus, the expression of either LMP1 or LMP2A may be sufficient to promote early-onset EBV-induced tumors in this model.IMPORTANCE EBV causes human lymphomas, but few models are available for dissecting how EBV causes lymphomas in vivo in the context of a host immune response. We recently used a newly developed cord blood-humanized mouse model to show that EBV can cooperate with human CD4 T cells to cause B cell lymphomas even when a major viral transforming protein, LMP1, is deleted. Here we examined whether the EBV protein LMP2A, which mimics B cell receptor signaling, is required for EBV-induced lymphomas in this model. We find that the deletion of LMP2A alone has little effect on the ability of EBV to cause lymphomas but delays tumor onset. The deletion of both LMP1 and LMP2A results in a smaller number of lymphomas in infected animals, with an even more delayed time to tumor onset. These results suggest that LMP1 and LMP2A collaborate to promote early-onset lymphomas in this model, but neither protein is absolutely essential.

Human Invariant NKT Cells Induce IL-1ß Secretion by Peripheral Blood Monocytes via a P2X7-Independent Pathway.

The cytokine IL-1ß plays a central role in inflammatory responses that are initiated by microbial challenges, as well as in those that are due to endogenous processes (often called sterile inflammation). IL-1ß secretion that occurs independently of microbial stimulation is typically associated with the presence of endogenous alarmins, such as extracellular ATP (an indicator of cytopathic damage). In this study, we show that IL-2-activated human invariant NKT (iNKT) cells stimulate the secretion of IL-1ß protein by human peripheral blood monocytes in a manner that requires neither the presence of microbial compounds nor signaling through the extracellular ATP receptor P2X7 Monocyte IL-1ß production was specifically induced by iNKT cells, because similarly activated polyclonal autologous T cells did not have this effect. Secretion of IL-1ß protein occurred rapidly (within 3-4 h) and required cell contact between the iNKT cells and monocytes. Similar to IL-1ß production induced by TLR stimulation, the iNKT-induced pathway appeared to entail a two-step process involving NF-κB signaling and IL1B gene transcription, as well as assembly of the NLRP3 inflammasome and activation of caspase-1. However, in contrast to the classical inflammasome-mediated pathway of IL-1ß production, activation of monocytes via P2X7 was dispensable for iNKT-induced IL-1ß secretion, and potassium efflux was not required. Moreover, the iNKT-induced effect involved caspase-8 activity, yet it induced little monocyte death. These results suggest that IL-2-activated human iNKT cells induce monocytes to produce IL-1ß through a distinctive pathway that does not require the presence of microbial danger signals or alarmins associated with cytopathic damage.

Salivary exRNA biomarkers to detect gingivitis and monitor disease regression.

AIM: This study tests the hypothesis that salivary extracellular RNA (exRNA) biomarkers can be developed for gingivitis detection and monitoring disease regression. MATERIALS AND METHODS: Salivary exRNA biomarker candidates were developed from a total of 100 gingivitis and non-gingivitis individuals using Affymetrix's expression microarrays. The top 10 differentially expressed exRNAs were tested in a clinical cohort to determine whether the discovered salivary exRNA markers for gingivitis were associated with clinical gingivitis and disease regression. For this purpose, unstimulated saliva was collected from 30 randomly selected gingivitis subjects, the gingival and plaque indexes scores were taken at baseline, 3 and 6 weeks and salivary exRNAs were assayed by means of reverse transcription quantitative polymerase chain reaction. RESULTS: Eight salivary exRNA biomarkers developed for gingivitis were statistically significantly changed over time, consistent with disease regression. A panel of four salivary exRNAs [SPRR1A, lnc-TET3-2:1, FAM25A, CRCT1] can detect gingivitis with a clinical performance of 0.91 area under the curve, with 71% sensitivity and 100% specificity. CONCLUSIONS: The clinical values of the developed salivary exRNA biomarkers are associated with gingivitis regression. They offer strong potential to be advanced for definitive validation and clinical laboratory development test.

Short- and Long-Term Effects of a Dentifrice Containing Dual Zinc plus Arginine on Intra-Oral Halitosis: Improvements in Breath Quality.

OBJECTIVES: These studies aimed to assess the short-term (12-hour, single use) and long-term (four weeks, continuous use) efficacy of a new Dual Zinc plus Arginine dentifrice against intra-oral halitosis versus a negative control. METHODS: Two clinical studies were conducted to assess the dentifrice: a four-week, continuous use parallel design versus a negative control and a single use crossover design versus a negative control. Both studies used organoleptic and hedonic odor judge scores measured 12 hours overnight after product use as the primary efficacy variable. Additionally, the single use study employed SIFT-MS to quantify the intra-oral concentration of volatile sulfur compounds as a complementary measure of efficacy. RESULTS: In both studies, the Dual Zinc plus Arginine dentifrice provided statistically significant improvements in breath quality across all measures versus a negative control. CONCLUSIONS: Improvements in breath quality were attributed to the effects of zinc cations delivered by the uniquely formulated dentifrice.

Enhanced In Vitro Zinc Bioavailability through Rational Design of a Dual Zinc plus Arginine Dentifrice.

OBJECTIVES: To investigate bioavailability enhancement of zinc on model oral surfaces and in oral biofilms in vitro through strategic formulation with two sources of zinc and L-arginine. METHODS: To modulate the bioavailability of active zinc ions in a zinc citrate dentifrice, an additive research strategy was pursued. A series of zinc citrate dentifrice formulations were prepared with increasing replacement of zinc citrate with zinc oxide (a water insoluble source of zinc ions) to generate a Dual Zinc active system. A screening of isolated zinc and amino acid effects in simple solutions using zeta potential and uptake to model oral surfaces was performed in an effort to determine the effect of particle charge on zinc bioavailability. Zinc delivery and antibacterial efficacy of the Dual Zinc plus Arginine dentifrice formula were tested using in vitro oral epithelial tissue and saliva-derived biofilm models. Furthermore, zinc penetration and retention were determined by subjecting in vitro biofilms to dynamic flow after treatment with the Dual Zinc plus Arginine dentifrice with treated biofilms evaluated for zinc using imaging mass spectrometry (I-MS). Bacterial adhesion to gingival epithelial cells treated with the Dual Zinc plus Arginine dentifrice was imaged upon challenging with Streptococcus gordonii. RESULTS: Addition of zinc oxide into a zinc citrate dentifrice formula enhanced the efficacy of the system against anaerobic biofilms in a concentration- dependent manner. L-arginine further provided a significant positive charge (+36 mV) to the zinc oxide suspension (+16 mV) as measured by zeta potential. Simple solutions of the Dual Zinc active showed increased zinc uptake on model oral surfaces as a direct function of L-arginine concentration. Antibacterial efficacy of a Dual Zinc plus Arginine dentifrice was evaluated through multiple mechanisms. Enhanced antibacterial performance was observed through significant reductions in metabolic activity as measured through bacterial glycolytic function (p = 0.0001) and total oxygen consumption (p = 0.0001). Greater penetration and retention of zinc was observed in bacterial biofilms treated with the Dual Zinc plus Arginine dentifrice in comparison to treatment with a Dual Zinc dentifrice after twelve hours of dynamic flow (10 mL/hour) in an in vitro drip flow biofilm culture. Confocal microscopy showed adherent bacteria on cheek cells treated with the Dual Zinc plus Arginine dentifrice formula. CONCLUSIONS: The combination of zinc citrate, zinc oxide, and the amino acid L-arginine in a dentifrice formula enhances the bioavailability of zinc to model oral tissue surfaces, resulting in unique physicochemical effects. The significant antimicrobial control associated with the Dual Zinc plus Arginine dentifrice provides a unique vehicle toward achieving whole mouth health.

Transition metal-catalyzed couplings of alkynes to 1,3-enynes: modern methods and synthetic applications.

The metal-catalyzed coupling of alkynes is a powerful method for the preparation of 1,3-enynes, compounds that are of broad interest in organic synthesis. Numerous strategies have been developed for the homo- and cross coupling of alkynes to enynes via transition metal catalysis. In such reactions, a major issue is the control of regio-, stereo-, and, where applicable, chemoselectivity. Herein, we highlight prominent methods for the selective synthesis of these valuable compounds. Further, we illustrate the utility of these processes through specific examples of their application in carbocycle, heterocycle, and natural product syntheses.

Synthesis of a 1,3-Bridged Macrobicyclic Enyne via Chemoselective Cycloisomerization Using Palladium-Catalyzed Alkyne-Alkyne Coupling.

A unique intramolecular Pd-catalyzed alkyne-alkyne coupling is presented. This transformation generates a strained, 1,3-bridged, macrocyclic enyne. The process was readily executed on gram scale, and the structure of the product was elucidated via X-ray crystallographic analysis. A mechanistic rationale for the observed chemoselectivity is provided.

A Water-Soluble Cationic Zinc Lysine Precursor for Coating ZnO on Biomaterial Surfaces.

A novel water-soluble cationic zinc lysine coordination compound, [Zn[(C6H14N2O2)]2Cl]Cl·2H2O (1), has been designed and synthesized and its crystal structure determined. The aqueous solution of this coordination compound is not only transparent and stable at room temperature but it is also nearly neutral (pH ∼ 7). It is worth noting that zinc oxide (ZnO) forms in situ upon dilution of a solution of the compound. The bioactivity of ZnO has been confirmed using an Alarma Blue assay. These unique properties allow the coordination compound to gently grow ZnO coating with excellent antibacterial benefits onto biomaterial surfaces in a facile and safe manner.