A Retrospective Case Series in Regenerative Endodontics: Trend Analysis Based on Clinical Evaluation and 2- and 3-dimensional Radiology

Introduction

A regenerative endodontic procedure (REP) is a biologically based treatment to functionally replace the pulp of infected immature permanent teeth. The purpose of this retrospective case series was to assess the outcome of REPs of infected immature permanent teeth in terms of periapical bone healing (PBH), root development (RD), and pulp vitality.

Loss of Tifab,a del(5q) MDS gene,alters hematopoiesis through derepression of Toll-like receptor–TRAF6 signaling

TRAF-interacting protein with forkhead-associated domain B (TIFAB) is a haploinsufficient gene in del(5q) myelodysplastic syndrome (MDS). Deletion of Tifab results in progressive bone marrow (BM) and blood defects, including skewed hematopoietic stem/progenitor cell (HSPC) proportions and altered myeloid differentiation. A subset of mice transplanted with Tifab knockout (KO) HSPCs develop a BM failure with neutrophil dysplasia and cytopenia. In competitive transplants, Tifab KO HSPCs are out-competed by wild-type (WT) cells, suggesting a cell-intrinsic defect. Gene expression analysis of Tifab KO HSPCs identified dysregulation of immune-related signatures, and hypersensitivity to TLR4 stimulation. TIFAB forms a complex with TRAF6, a mediator of immune signaling, and reduces TRAF6 protein stability by a lysosome-dependent mechanism. In contrast, TIFAB loss increases TRAF6 protein and the dynamic range of TLR4 signaling, contributing to ineffective hematopoiesis. Moreover, combined deletion of TIFAB and miR-146a, two genes associated with del(5q) MDS/AML, results in a cooperative increase in TRAF6 expression and hematopoietic dysfunction. Re-expression of TIFAB in del(5q) MDS/AML cells results in attenuated TLR4 signaling and reduced viability. These findings underscore the importance of efficient regulation of innate immune/TRAF6 signaling within HSPCs by TIFAB, and its cooperation with miR-146a as it relates to the pathogenesis of hematopoietic malignancies, such as del(5q) MDS/AML.Myelodysplastic syndrome (MDS) refers to a group of hematopoietic stem cell (HSC) disorders associated with ineffective hematopoiesis, blood cytopenias, myeloid dysplasia, genomic instability, and predisposition to acute myeloid leukemia (AML) or bone marrow failure (BMF). Recent classifications of MDS describe eight subtypes based on biological, genetic, and morphological features (Cazzola and Malcovati, 2010). Independent of classification, MDS is propagated by rare and defective HSCs, and is defined by recurring cytogenetic changes and somatic point mutations. The most common cytogenetic alteration in MDS is deletion of chromosome (chr) 5q (del(5q)). In the absence of other cytogenetic alterations, MDS patients with a del(5q) exhibit refractory anemia, neutropenia, and elevated platelets associated with hypolobulated megakaryocytes (Giagounidis et al., 2006). Del(5q) is found in 25% of therapy-related MDS cases, which occur as a result of treatment with alkylating agents for unrelated conditions, and is strongly correlated with progression to AML (Haase et al., 2007; Haase, 2008; Qian et al., 2010). Although two commonly deleted regions (CDRs) have been mapped on chr 5q each spanning ∼1 Mb, a distal locus at 5q33.1, and a proximal locus at 5q31.1(Zhao et al., 1997), there are multiple genes that likely contribute to the pathogenesis of del(5q) MDS (Le Beau et al., 1989; Willman et al., 1993; Boultwood et al., 1994, 1997, 2000, 2002, 2007; Jaju et al., 1998). Clonal dominance of del(5q) cells is driven by haploid expression of CSNK1A1 (Schneider et al., 2014), whereas the erythroid defect has been attributed to RPS14 genes located within the distal CDR (Ebert et al., 2008; Barlow et al., 2010). Thrombocytosis associated with megakaryocytic dysplasia, neutropenia, and clonal dominance, are caused by loss of two miRNAs, miR-145 and miR-146a, in del(5q) MDS patients (Kumar et al., 2009; Starczynowski et al., 2010). Germline knockout of mouse miR-146a results in an early onset of myeloid expansion in the BM, and progression to more aggressive diseases such as lymphomas, BMF, and myeloid leukemia (Lu et al., 2010; Boldin et al., 2011; Zhao et al., 2011). Furthermore, overexpression of TRAF6, a miR-146a target gene, in mouse HSPCs mimics certain hematopoietic defects observed in miR-146a–deficient mice, including neutropenia, dysplasia, and myeloid leukemia. Overexpression of TRAF6, however, also results in elevated platelets. Some of the effects are mediated by IL-6, as overexpression of TRAF6 in Il6-deficient HSPCs restores platelets and neutrophil counts. DIAPH1, which encodes mDia1, is located on 5q31.3, which resides between the two commonly deleted regions in del(5q) MDS. mDia1-deficient mice exhibit an age-dependent granulocytopenia, and myeloid dysplasia in the BM, in part through increased TLR4 signaling. The proximal CDR at 5q31.1 is associated with aggressive forms of del(5q) and includes ∼12 coding genes. Two of these genes, CTNNA1 and EGR1, are thought to contribute to cell survival and myeloproliferation, respectively (Joslin et al., 2007; Liu et al., 2007). HSPA9 and PPP2CA have also been implicated in aspects of the del(5q) MDS/AML phenotype. Despite this recent progress, detailed molecular, cellular, and genetic analyses of candidate genes on chr 5q are required to completely understand the pathogenesis of del(5q) MDS/AML.Derepression of TRAF6, as a result of miR-146a haploinsufficiency, is one molecular consequence of del(5q) (Starczynowski et al., 2010; Boldin et al., 2011). TRAF6 is an E3 ubiquitin ligase and signal transducer of the innate immune signaling pathway in response to pathogens and host damage-associated molecules (Wu and Arron, 2003). A search of annotated genes within or near the CDR in del(5q) revealed a relatively uncharacterized gene, TRAF-interacting protein with forkhead-associated domain B (TIFAB). TIFAB resides within the proximal CDR on band 5q31.1, and belongs to a family of forkhead-associated domain proteins that also includes TIFA. TIFA was originally identified as a TRAF6-interacting protein in a yeast two-hybrid screen (Kanamori et al., 2002; Takatsuna et al., 2003), whereas TIFAB was identified as a TIFA-related protein by an in silico homology screen (Matsumura et al., 2004, 2009). To investigate whether loss of TIFAB is important to the pathophysiology of del(5q) MDS/AML, in this study, we characterized a germline Tifab knockout (KO) mouse. Tifab^−/− mice exhibit progressive hematopoietic defects, including skewed HSPC proportions, altered myeloid differentiation, and a BMF-like disease associated with BM dysplasia and cytopenia. Tifab^−/− BM cells are hypersensitive to Toll-like receptor 4 (TLR4) stimulation, suggesting that loss of TIFAB alters the innate immune pathway. Independent of TRAF6 mRNA, TIFAB loss results in stabilization of TRAF6 protein. Moreover, combined deletion of TIFAB and miR-146a results in a cooperative increase in TRAF6 expression and hematopoietic dysfunction in vivo. This provides a potential molecular explanation for altered TLR4 sensitivity and the BMF phenotype. Collectively, our results provide evidence that deletion of TIFAB contributes to an MDS-like hematopoietic phenotype in mice by changing the dynamic range of the innate immune pathway in HSPCs through the regulation of TRAF6 protein stability.     

Effect of resin-modified glass-ionomer cement lining and composite layering technique on the adhesive interface of lateral wall

Interface integrity can be maintained by setting the composite in a layering technique and using liners.

Objective

The aim of this in vitro study was to verify the effect of resin-modified glass-ionomer cement (RMGIC) lining and composite layering technique on the bond strength of the dentin/resin adhesive interface of lateral walls of occlusal restorations.

Material and Methods

Occlusal cavities were prepared in 52 extracted sound human molars, randomly assigned into 4 groups: Group 2H (control) – no lining + two horizontal layers; Group 4O: no lining + four oblique layers; Group V-2H: RMGIC lining (Vitrebond) + two horizontal layers; and Group V-4O: RMGIC lining (Vitrebond) + four oblique layers. Resin composite (Filtek Z250, 3M ESPE) was placed after application of an adhesive system (Adper™ Single Bond 2, 3M ESPE) dyed with a fluorescent reagent (Rhodamine B) to allow confocal microscopy analysis. The teeth were stored in deionized water at 37^oC for 24 hours before being sectioned into 0.8 mm slices. One slice of each tooth was randomly selected for Confocal Laser Scanning Microscopy (CLSM) analysis. The other slices were sectioned into 0.8 mm x 0.8 mm sticks to microtensile bond strength test (MPa). Data were analyzed by two-way ANOVA and Fisher’s test.

Results

There was no statistical difference on bond strength among groups (p>0.05). CLSM analysis showed no significant statistical difference regarding the presence of gap at the interface dentin/resin among groups.

Conclusions

RMGIC lining and composite layering techniques showed no effect on the microtensile bond strength and gap formation at the adhesive interface of lateral walls of high C-factor occlusal restorations.     

Mortality in patients with ankylosing spondylitis in Argentina

Some reports describe an increased mortality in patients with ankylosing spondylitis (AS) compared to the general population. The aims of this study were to evaluate the cumulative survival in patients with AS and to establish possible factors associated with mortality. In cross-sectional retrospective study, AS patients were included according to 1984 modified NY criteria, in the 2000–2010 period, the prevalence of mortality was determined by review of medical records, telephone contact, family reports, and death certificates, and it was compared with mortality in Argentina’s general population. One hundred twenty-seven patients were studied, 96 (75.6 %) were male, median age 49 years (interquartile range (IQR) 34–60) and median disease duration 8 years (IQR 4–17). During the follow-up period, 9 patients died (7.1 %). The median estimated survival from diagnosis of AS was 39 years (IQR 34–50) and median cumulative survival was 76 years (IQR 74–85). Cardiovascular disease was the most frequent cause of death (5/9 patients). Deceased patients had a mean age and a mean AS disease duration significantly higher than living patients (68.1?±?12.4 years vs 46.4?±?15.09 years, p?=?0.0001 and 33?±?13.7 years vs 12?±?10.7 years, p?=?0.001, respectively), higher frequency of total surgeries [3/5 (60 %) vs 5/105 (4.76 %), p?=?0.002] and cauda equina syndrome [3/6 (50 %) vs 2/116 (1.72 %), p?=?0.001], respectively. Frequency of mortality in AS patients was higher than the crude mortality rate of Argentina’s general population in the same period, with cardiovascular cause being the most frequent one.     

Blockade of CD40–TRAF2,3 or CD40–TRAF6 is sufficient to inhibit pro‐inflammatory responses in non‐haematopoietic cells

Inhibition of the CD40–CD154 pathway controls inflammatory disorders. Unfortunately, administration of anti‐CD154 monoclonal antibodies causes thromboembolism. Blockade of signalling downstream of CD40 may represent an approach to treat CD40‐driven inflammatory disorders. Blocking tumour necrosis factor receptor‐associated factor 6 (TRAF6) signalling downstream of CD40 in MHC II⁺ cells diminishes inflammation. However, CD40–TRAF6 blockade may cause immunosuppression. We examined the role of CD40–TRAF2,3 and CD40–TRAF6 signalling in the development of pro‐inflammatory responses in human non‐haematopoietic and monocytic cells. Human aortic endothelial cells, aortic smooth muscle cells, renal proximal tubule epithelial cells, renal mesangial cells and monocytic cells were transduced with retroviral vectors that encode wild‐type CD40, CD40 with a mutation that prevents TRAF2,3 recruitment (ΔT2,3), TRAF6 recruitment (ΔT6) or both TRAF2,3 plus TRAF6 recruitment (ΔT2,3,6). Non‐haematopoietic cells that expressed CD40 ΔT2,3 exhibited marked inhibition in CD154‐induced up‐regulation of vascular cell adhesion molecule 1, intercellular adhesion molecule 1 (ICAM‐1), monocyte chemotactic protein 1 (MCP‐1), tissue factor and matrix metalloproteinase 9. Similar results were obtained with cells that expressed CD40 ΔT6. Although both mutations impaired ICAM‐1 up‐regulation in monocytic cells, only expression of CD40 ΔT6 reduced MCP‐1 and tissue factor up‐regulation in these cells. Treatment of endothelial and smooth muscle cells with cell‐permeable peptides that block CD40–TRAF2,3 or CD40–TRAF6 signalling impaired pro‐inflammatory responses. In contrast, while the CD40–TRAF2,3 blocking peptide did not reduce CD154‐induced dendritic cell maturation, the CD40–TRAF6 blocking peptide impaired this response. Hence, preventing CD40–TRAF2,3 or CD40–TRAF6 interaction inhibits pro‐inflammatory responses in human non‐haematopoietic cells. In contrast to inhibition of CD40–TRAF6 signalling, inhibition of CD40–TRAF2,3 signalling did not impair dendritic cell maturation. Blocking CD40–TRAF2,3 signalling may control CD40–CD154‐dependent inflammatory disorders.     

Commentary: A Practical Guide for Translating Basic Research on Affective Science to Implementing Physiology in Clinical Child and Adolescent Assessments

The National Institute of Mental Health recently launched the Research Domain Criteria (RDoC). RDoC is a framework that facilitates the dimensional assessment and classification of processes relevant to mental health (e.g., affect, regulation, cognition, social affiliation), as reflected in measurements across multiple units of analysis (e.g., physiology, circuitry, genes, self-reports). A key focus of RDoC involves opening new lines of research examining patients’ responses on biological measures, with the key goal of developing new therapeutic techniques that effectively target mechanisms of mental disorders. Yet applied researchers and practitioners rarely use biological measures within mental health assessments, which may present challenges in translating RDoC-guided research into improvements in patient care. Thus, if RDoC is to result in research that yields clinical tools that reduce the burden of mental illness and improve public health, we ought to develop strategies for effectively implementing biological measures in the context of clinical assessments. In this special issue, we sought to provide an initial step in this direction by assembling a collection of articles from leading research teams carrying out pioneering work on implementing multimodal assessments (biological, subjective, behavioral) of affective processes in applied settings. In this commentary, we expand upon the work presented in this special issue by making a series of suggestions for how to most parsimoniously conduct multimodal assessments of affective processes in applied research and clinical settings. We hope that this approach will facilitate translations of the RDoC framework into applied research and clinic settings.     

Evaluation of correlation of articular cartilage staining for DDR2 and proteoglycans with histological tissue damage and the results of radiographic assessment in patients with early stages of knee osteoarthritis

Objective: To determine, if staining of articular cartilage for proteoglycans (natural element of healthy and functioning cartilage) and discoidin domain receptor 2 (DDR2) (a protein associated with articular cartilage degradation) is correlated with histological tissue damage or radiographic assessment score in patients with early stages of knee osteoarthritis (OA). Method: 40 patients, with early stage OA were enrolled, from whom the biopsies for histological and immunohistochemical studies were obtained from edge of the femoral condyle during the arthroscopy. Semi-quantitative computer based analysis was used to evaluate the proportion of staining in histological sections. Results: No correlation was shown between the proportion of tissue stained for DDR2 and histological score or the results of radiographic assessment of tibiofemoral (TF) joint. There was a negative correlation between the proportion of tissue stained for DDR2 and radiographic grade of patellofemoral (PF) OA (Spearman r=-0.34; 95% CI -0.60 to -0.02; P=0.03). No correlation was shown between the proportion of tissue stained for proteoglycans and histological score or the results of radiographic assessment of TF and PF joints. A negative correlation was found between proportion of tissue stained for DDR2 and proteoglycans. Spearman r=-0.43; 95% CI=-0.66 to -0.12; P=0.006. Conclusion: Production of DDR2 in articular cartilage could be related to early stages of OA, as it is significantly correlated to decrease of staining for cartilage proteoglycans. The role of production of DDR2 in cartilage may be decreased in stages, where higher grades of OA are detected on the radiographs.     

Comprehensive elucidation of amino acid profile in human follicular fluid and plasma of in vitro fertilization patients

Abstract

The assessment of oocyte quality in human in vitro fertilization (IVF) is in focus of intensive studies. Follicular fluid (FF) constitutes the major medium for the developing oocyte and therefore it is a perfect target to search for the biomarkers of female infertility. The objective of this study was to compare the amino acid (AA) profile of human FF and plasma (PL) (from 96 IVF patients) and to examine if the AA composition is related to oocyte quality, IVF results and women’s infertility. In both biological fluids, Gln, Gly and Ala appeared as dominant AAs. Most of AAs are more abundant in PL; the exceptions are Glu, Thr, Ala and Gly, which are higher in FF and Gln, Arg and Phe, the contents of which are similar in both biological fluids. Ser in FF and Met and Phe in PL were detected as potential biomarkers as their content varied depending on the IVF outcome. Significant differences were also detected between the groups of different infertility reasons. Our results suggest that intra-follicular AAs might reflect the condition of the preovulatory follicle and together with PL, AAs can be used to characterize the infertility etiology and oocyte quality related to IVF outcome.