HHV-6 cell receptor CD46 expression on various cell subsets of six blood and graft sources: A prospective series

BackgroundCord Blood (CB) are increasingly used as an alternative stem cells source in adults for allogeneic Stem Cell Transplantation (allo-SCT). The risk of human herpesvirus (HHV-6) reactivation is significantly higher after CB transplant vs unrelated peripheral blood stem cells (PBSC) allo-SCT. Higher HHV-6 cell receptor CD46 expression on progenitor cells in CB may explain this difference.ObjectivesTo prospectively compare the HHV-6 cell receptor CD46 expression on various cell subsets of three freshly harvested blood sources on one hand and of three graft sources on the other hand.Study design52 samples were used for the purpose of this study. They were issued from peripheral blood (PB, n = 10), G-CSF mobilised PB (GCSF-PB, n = 10), cord blood (CB, n = 10), unmanipulated bone marrow (uBM, n = 5), leukapheresis product (LP, n = 10) and thawed CB graft (n = 7). CD46 expression was assessed by FACS analysis on total lymphocytes, monocytes, NK cells, T and B cells subsets, plasmacytoid (pDCs) dendritic cells and stem cells.ResultsAs all cell subsets were found CD46 positive, CD46 mean fluorescence intensity (MFI) was then considered for comparison between the three blood sources and the three graft sources. The most impressive result observed was that HHV-6 cell receptor CD46 expression was significantly reduced in almost all cell components of thawed CB graft compared to other graft sources.ConclusionsThis original study shows strong differences in term of quantitative CD46 expression between several blood and grafts samples. Our results suggest that other factors than the qualitative CD46 expression play a role in the higher HHV-6 reactivation observed after CB transplant in adults.     

Removal of osseointegrated dental implants: a systematic review of explantation techniques

Objectives

This systematic review aims to evaluate current literature regarding available techniques for removal of osseointegrated implants in terms of explantation’s success, complications, and bone loss.

Material and methods

Two reviewers conducted a systematic literature search through electronic databases (PubMed and EMBASE), complimented by manual and grey literature searches. Successful explantation was defined as the primary outcome. Complications and availability of residual bone for immediate implantation were defined as secondary outcomes.

Results

Eighteen articles, comprising 372 implants and 241 patients, were included. Five techniques were identified: reverse torque, trephines, burs, piezosurgery, and laser-assisted explantation. Peri-implantitis was the most common reason for explantation, followed by crestal bone loss, fracture, and malpositioning. The reverse torque was the most frequently reported technique (284 implants) with 87.7% success rate. Burs were used for explantation of 49 implants with a 100% success rate, while trephines were utilized for removal of 35 implants with 94% success. Piezosurgery (11 implants) and Er.Cr:YSGG laser (1 implant) showed 100% success. One study reported perforation of the sinus floor following trephine explantation, while another reported fracture of 3 implants following reverse torque application. Further analysis was hindered by the quality of the available studies and their lack of data.

Conclusions

Reverse torque seems the most conservative, and in the authors’ opinion, should be the first choice for explantation despite its inferior success rate. Additional studies with randomized controlled designs and larger sample sizes are required.

Clinical relevance

Dental implants have become the leading choice to replace missing teeth with gradually increasing numbers of complications and failures. An effective, conservative, and economic explantation technique is necessary to allow a successive implant placement.

     

Ischemia Modified Albumin for the assessment of patients presenting to the emergency department with acute chest pain but normal or non-diagnostic 12-lead electrocardiograms and negative cardiac troponin T

BACKGROUND: The diagnosis of myocardial ischemia in patients with acute chest pain at rest but non-diagnostic electrocardiograms (ECG) is problematic. Ischemia Modified Albumin (IMA) is a new biochemical marker of ischemia, which may be useful to characterise acute coronary syndrome (ACS) patients. METHODS: We studied 131 patients (mean age 58.5 years; 95 male) presenting to the emergency department with symptoms suggestive of ACS but with normal or non-diagnostic ECGs. Cardiac troponin T (cTnT) and IMA were measured within 3 h of last chest pain episode. Based on hospital diagnostic test results, patients were classified as having ACS or non-ischemic chest pain (NICP), by two independent cardiologists unaware of IMA results. RESULTS: Mean IMA levels (U/ml) were higher in patients with ACS (98.3+/-11) compared to patients with NICP (85.5+/-15); p<0.0001. IMA levels >93.5 U/ml demonstrated a sensitivity and specificity of 75% for the diagnosis of ACS; area under the receiver operator characteristic curve 0.78 (95% CI: 0.70-0.85). If we applied the manufacturer cutoff point of 85 U/ml, the sensitivity of IMA increased to 90.6% with a specificity of 49.3% (negative predictive value=84.6%). In combination with cTnT (6-12 h) (>0.05 ng/ml), the sensitivity increased to 92.2%. After multivariate analysis, IMA levels >85 U/ml (odds ratio=14.6 [95% CI 4.4-48.4]; p<0.0001), age and prior myocardial infarction were independent predictors of ACS. CONCLUSION: IMA may be a useful biomarker for the identification of ACS in patients presenting with typical acute chest pain but normal or non-diagnostic ECGs.     

Cdc42 is a key regulator of B cell differentiation and is required for antiviral humoral immunity

The small Rho GTPase Cdc42, known to interact with Wiskott–Aldrich syndrome (WAS) protein, is an important regulator of actin remodeling. Here, we show that genetic ablation of Cdc42 exclusively in the B cell lineage is sufficient to render mice unable to mount antibody responses. Indeed Cdc42-deficient mice are incapable of forming germinal centers or generating plasma B cells upon either viral infection or immunization. Such severe immune deficiency is caused by multiple and profound B cell abnormalities, including early blocks during B cell development; impaired antigen-driven BCR signaling and actin remodeling; defective antigen presentation and in vivo interaction with T cells; and a severe B cell–intrinsic block in plasma cell differentiation. Thus, our study presents a new perspective on Cdc42 as key regulator of B cell physiology.B cells provide a critical line of defense from pathogenic infections through the production of highly specific antibodies. The initial stages of B cell development occur in the bone marrow, where hematopoietic stem cells undergo stepwise rearrangements of the genes encoding the B cell receptor (BCR) and changes in the expression of cell surface receptors (Hardy et al., 1991). Immature B cells egress the bone marrow and migrate to the spleen to complete their development, going through transitional stages. Mature follicular B cells then recirculate throughout the body in search for cognate antigen, continually entering secondary lymphoid organs, including the LNs and spleen. Specific recognition of antigen by the BCR provides the first signal required for B cell activation. Typically, a second signal is required for maximal activation and is provided by CD4⁺ helper T cells after the presentation of processed antigen on the B cell surface. These two signals in combination trigger the proliferation and differentiation of B cells, which go on to form antibody-secreting plasma cells and to establish germinal center responses for affinity maturation (Rajewsky, 1996).B cell activation in vivo is predominantly triggered by antigen on the surface of a presenting cell (Batista and Harwood, 2009). The prevalence of this mode of activation has brought about a reevaluation of the importance of the cytoskeleton, given that the recognition of tethered antigen requires considerable alteration in B cell morphology (Fleire et al., 2006). Antigen-induced BCR signaling leads to radical reorganization of the actin cytoskeleton resulting in the modification of the BCR dynamics at the cell surface (Hao and August, 2005; Treanor et al., 2010; Treanor et al., 2011). Moreover the binding of membrane-bound antigen to cognate BCR triggers a cascade of intracellular signaling events that induces actin-dependent spreading of the B cell across the antigen-containing surface (Weber et al., 2008; Sohn et al., 2008; Depoil et al., 2008). However the mediators that link BCR signaling with reorganization of the actin cytoskeleton are currently not well defined.Among actin regulators, the RhoGTPases are a highly conserved family that function as molecular switches by cycling between inactive GDP (guanosine diphosphate) and active GTP (guanosine triphosphate) bound states (Tybulewicz and Henderson, 2009). RhoGTPase activity is modulated by G-nucleotide exchange factors (GEF) that promote the formation of the GTP-bound state and binding to various effectors involved in actin reorganization. Conversely, GTPase-activating proteins (GAP) catalyze the hydrolysis of GTP and thereby switch off RhoGTPase activity. The importance of the RhoGTPases as a whole in the regulation of B cell responses is highlighted by the far-reaching consequences that impaired activity of several GEFs, such as Vav and DOCK8, has on humoral immune responses (Doody et al., 2001; Fujikawa et al., 2003; Randall et al., 2009; Zhang et al., 2009).The importance of Rho GTPases in B cell physiology has been well established. For example, RhoA has been shown to regulate BCR signaling by influencing inositol-3 phosphate synthesis and calcium signaling (Saci and Carpenter, 2005). Moreover, B cell–specific inactivation of both Rac1 and Rac2 leads to virtually complete absence of B cells (Walmsley et al., 2003), and inactivation of Rac1 results in defects in spreading in transitional cells (Brezski and Monroe, 2007). However, although the inactivation of Rac2 leads to defects in B cell adhesion and synapse formation, it is unclear whether these proteins are involved in actin-dependent spreading in mature B cells (Arana et al., 2008).Cdc42 has been little characterized in B cells, in spite of its proven chief role as an essential regulator of cell cycle (Johnson and Pringle, 1990), cell polarity (Etienne-Manneville, 2004), and actin cytoskeleton in other cellular systems. This is likely due, at least in part, to the reported mild phenotype of mice lacking Cdc42 in B cells (Guo et al., 2009) compared with the severe deficiencies observed in animals lacking Rac family members (Walmsley et al., 2003). However, the mild phenotype is somehow surprising given that Cdc42 directly or indirectly associates with Wiskott–Aldrich Syndrome Protein (WASp) and in complex with Arp2/3 regulates cytoskeleton remodeling (Symons et al., 1996; Aspenström et al., 1996; Kolluri et al., 1996). Importantly, mutations in WAS gene lead to a X-linked, recessive disease characterized by recurrent infections, abnormal lymphocyte function, as well as an increased risk for systemic autoimmunity (Derry et al., 1994; Sullivan et al., 1994). WASp deficient B cells play a primary role in driving autoimmunity (Becker-Herman et al., 2011). The Cdc42 effectors WASp and N-WASp have both been implicated the regulation of actin reorganization in response to BCR antigen engagement (Westerberg et al., 2012; Liu et al., 2013). Besides, expression of a dominant negative form of Cdc42 in B cells leads to alterations of the actin cytoskeleton (Westerberg et al., 2001). In addition, Cdc42 has been shown to play a role in the polarization and secretion of lysosomal protein involved in antigen extraction (Yuseff et al., 2011).Here, we used a strategy harnessing the mb1 promoter to generate mice with a selective and very effective deletion of Cdc42 in early B cell progenitors (Hobeika et al., 2006). Using this model, we demonstrated that Cdc42 plays an essential role in many aspects of B cell biology, including the formation of mature B cells and the establishment of antibody responses. We went on to dissect the underlying cause of the severe immunodeficiency of these mice and found that Cdc42-deficient B cells exhibit defects in BCR signaling and presentation of internalized antigen, leading to reduced B–T cell interactions and the absence of germinal center responses in vivo. Moreover, Cdc42-deficient B cells can normally proliferate and class switch when stimulated with CD40 or LPS, but they are completely impaired in their ability to differentiate into plasma cells. Together, these attributes render Cdc42-deficient mice unable to mount antibody responses after immunization with model antigen or viral infection, and highlight a fundamental role for this RhoGTPase in the regulation of B cell responses.     

Laboratory Biosafety: Benchmarking public health pain management practices during school immunizations

BackgroundPain and fear during immunizations can affect children and their future behaviour toward immunization. These negative experiences can be amplified when children receive vaccines as part of school-based immunization programs, where parental or tutor supports are missing. In 2015, HELPinKIDS&ADULTS, a Canadian network of experts, published a clinical practice guideline (CPG) on the management of pain and fear during immunization. This guideline has been endorsed by international, national and provincial organizations. However, the level of integration and implementation of the CPG into local and community immunization programs such as school-based immunization clinics is unclear.MethodsAn investigation whether public health units in Ontario integrated and implemented the pain and fear interventions recommended by the CPG into school-based immunization policies and practices was concluded.ResultsThe study shows that the majority of public health units do have pain and fear policies and procedures in place, but interventions are not integrated in a consistent and formal manner, leading to suboptimal uptake of interventions during immunizations at school.ConclusionFor pain interventions to be applied with sufficient fidelity and in enough individuals to have a meaningful effect, organizational leaders need to create directives and procedures that support implementation in a systematic and accountable manner.     

Active chi-like sequences are present in the ITS1 region of polyembryonic adult Collyriclum faba trematodes encysted in pairs

Collyriclum faba (Plagiochiida: Collyriclidae) adults occur in pairs within subcutaneous cysts. Here, we tested the extensive C. faba infrapopulation for five DNA loci known to display variability among Central European C. faba individuals. The infrapopulation tested shared 100 % similarity in four of the five mitochondrial and nuclear DNA loci tested. Contrariwise, the internal transcribed spacer 1 (ITS1) loci in all but one individual differed from each other. We found only 0.0–1.5 base substitutions per 1,000 sites within the cysts, while we found 0.7–9.0 substitutions between the cysts of the single host and 3.0–9.0 substitutions when comparing C. faba individuals isolated from different host individuals. We observed the most of the ITS1 variability within 48 bp repetitive sequences featured by the chi-like sequence 5′-GCTTGTCTGCC-3′ at their beginning. Similarly to the extensive C. faba infrapopulation examined, we determined the presence of highly variable number of repetitive sequences within the ITS1 locus of C. faba isolated from multiple host species and from various geographic locations. While similar variability was observed earlier in mutually unrelated specimens of several Schistosomatidae and Microphallidae species, here, we for the first time document it among multiple individuals of a single infracommunity possessing single mitochondrial haplotype. Lower ITS1 evolutionary divergence rates observed between individuals within the cysts when compared to those between the cysts suggest that the recombination occurs at multiple stages of the life cycle. We propose DNA recombination involving chi-like sequences to serve as a general feature shared by multiple families of digenetic trematodes to increase genetic diversity of their polyembryonic populations infecting their definitive hosts.     

Increased myofilament Ca2+ sensitivity and diastolic dysfunction as early consequences of Mybpc3 mutation in heterozygous knock-in mice

Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in MYBPC3 encoding cardiac myosin-binding protein C (cMyBP-C). The mechanisms leading from gene mutations to the HCM phenotype remain incompletely understood, partially because current mouse models of HCM do not faithfully reflect the human situation and early hypertrophy confounds the interpretation of functional alterations. The goal of this study was to evaluate whether myofilament Ca(2+) sensitization and diastolic dysfunction are associated or precede the development of left ventricular hypertrophy (LVH) in HCM. We evaluated the function of skinned and intact cardiac myocytes, as well as the intact heart in a recently developed Mybpc3-targeted knock-in mouse model carrying a point mutation frequently associated with HCM. Compared to wild-type, 10-week old homozygous knock-in mice exhibited i) higher myofilament Ca(2+) sensitivity in skinned ventricular trabeculae, ii) lower diastolic sarcomere length, and faster Ca(2+) transient decay in intact myocytes, and iii) LVH, reduced fractional shortening, lower E/A and E'/A', and higher E/E' ratios by echocardiography and Doppler analysis, suggesting systolic and diastolic dysfunction. In contrast, heterozygous knock-in mice, which mimic the human HCM situation, did not exhibit LVH or systolic dysfunction, but exhibited higher myofilament Ca(2+) sensitivity, faster Ca(2+) transient decay, and diastolic dysfunction. These data demonstrate that myofilament Ca(2+) sensitization and diastolic dysfunction are early phenotypic consequences of Mybpc3 mutations independent of LVH. The accelerated Ca(2+) transients point to compensatory mechanisms directed towards normalization of relaxation. We propose that HCM is a model for diastolic heart failure and this mouse model could be valuable in studying mechanisms and treatment modalities.     

Interactions between glucocorticoids and warfarin in chronic inflammatory (autoimmune) diseases

Glucocorticosteroids are still very important part of the treatment of chronic inflammatory disorders. Their use is often accompanied by unpleasant adverse effects, problems associated with withdrawal during their long-term use and interactions with concomitantly administered drugs. One of the important interactions that may often occur in clinical practice is interaction with warfarin. Despite the fact that as glucocorticosteroids so warfarin are used for many years and seem to be completely known, their co-administration is still accompanied by uncertainties. The interaction may have pharmacodynamic or pharmacokinetic character and both types result in increased risk of bleeding. The pharmacodynamic interaction can be expected to increase a risk of gastrointestinal bleeding to which a gastrotoxicity of glucocorticosteroids contributes. A pharmacokinetic interaction is considered to influence a hepatic metabolism of warfarin and to increase its availability. The exact mechanism is still not fully understood. Manifestations of both types of interactions are taken up with a delay. Co-administration requires increased attention and close monitoring of international normalized ratio. At higher doses of glucocorticosteroids proton pump inhibitors are also effective in prevention of gastrotoxicity.