Role of Fractalkine-CX3CR1 Axis in Acute Rejection of Mouse Heart Allografts Subjected to Ischemia Reperfusion Injury.

Transplantation outcomes are affected by the increase in rejection associated with ischemia reperfusion injury (IRI). Fractalkine (FKN), a chemokine for recruitment of CX3CR1+ leukocytes, contributes to the pathogenesis of various inflammatory diseases. Herein, we evaluated the importance of the FKN-CX3CR1 axis during IRI-related rejections using a mouse heterotopic heart transplantation model. FKN expression and graft survival was compared between wild-type C57BL/6 recipients transplanted with BALB/c hearts preserved for 8 (WT-IRI) and 0.5 h (WT-control) at 4°C. Graft survival of WT-IRI was shorter than that of WT-control. FKN was expressed on the vascular endothelium in WT-IRI allografts, but minimally in WT-control. The role of the FKN-CX3CR1 axis in IRI-related rejection was directly investigated using the transplant model with CX3CR1-deficient recipients (CX3CR1 KO-IRI) or treatment with anti-mouse FKN monoclonal antibodies. Graft survival of CX3CR1 KO-IRI was longer than that of WT-IRI; antibody treatment prolonged graft survival. The contribution of CX3CR1+ monocytes to IRI-related rejection was evaluated by adoptive transfer to CX3CR1 KO-IRI. Adoptive transfer of CX3CR1+ monocytes attenuated the effect of prolonged graft survival in CX3CR1 KO-IRI. Overall, the FKN-CX3CR1 axis plays a major role during IRI-related rejection; its blockade has the potential to improve the outcomes of deceased donor transplantation.

Comparison of torque, force generation and canal shaping ability between manual and nickel-titanium glide path instruments in rotary and optimum glide path motion.

This study aimed to analyze force/torque generation and canal volume changes of NiTi rotary glide path preparation using HyFlex EDM Glide Path File in comparison to manual stainless steel K-file instrumentation. Thirty extracted mandibular incisors with a minimally curved and narrow root canal were randomly divided into three groups (n = 10) according to the instrumentation kinematics: Optimum Glide Path motion (OGP) or continuous rotation (CR) with HyFlex EDM Glide Path Files using a custom-made automated-root-canal-preparation device and manual instrumentation with stainless steel K-files (SS) in watch-winding motion. Torque and force were monitored with a custom-made torque/force analyzing device. Canal volume changes and transportation values were measured on micro-computed tomographic images taken before and after the glide path preparation. The data were statistically evaluated using Kruskal-Wallis test and Mann-Whitney U test with Bonferroni correction, with a significance level set at 5%. Maximum upward apical force, representing the screw-in force, was lower in groups OGP and CR compared with that in group SS (P < 0.05). Group CR showed the highest maximum clockwise torque value and canal volume changes, followed by groups OGP and SS (P < 0.05). Canal transportation values at 1 and 3 mm from the apex were not significantly different among groups. Within the limitations of this study, rotary glide path preparation generated smaller screw-in force, larger torque and larger canal volume changes than manual preparation. OGP motion generated smaller torque and less canal volume changes than CR.

Unveiling Spatial Immune Cell Profile in Kidney Allograft Rejections Using 36-plex Immunofluorescence Imaging.

BACKGROUND: Kidney allograft rejections are orchestrated by a variety of immune cells. Because of the complex histopathologic features, accurate pathological diagnosis poses challenges even for expert pathologists. The objective of this study was to unveil novel spatial indices associated with transplant rejection by using a spatial bioinformatic approach using 36-plex immunofluorescence image data. METHODS: The image obtained from 11 T cell-mediated rejection (TCMR) and 12 antibody-mediated rejection (AMR) samples were segmented into 753 737 single cells using DeepCell's Mesmer algorithm. These cells were categorized into 13 distinct cell types through unsupervised clustering based on their biomarker expression profiles. Cell neighborhood analysis allowed us to stratify kidney tissue into 8 distinct neighborhood components consisting of unique cell type enrichment profiles. RESULTS: In contrast to TCMR samples, AMR samples exhibited a higher frequency of neighborhood components that were characterized by an enrichment of CD31+ endothelial cells. Although the overall frequency of CD68+ macrophages in AMR samples was not significantly high, CD68+ macrophages within endothelial cell-rich lesions exhibited a significantly higher frequency in AMR samples than TCMR samples. Furthermore, the frequency of interactions between CD31+ cells and CD68+ cells was significantly increased in AMR samples, implying the pivotal role of macrophages in AMR pathogenesis. Importantly, patients demonstrating a high frequency of CD31:CD68 interactions experienced significantly poorer outcomes in terms of chronic AMR progression. CONCLUSIONS: Collectively, these data indicate the potential of spatial bioinformatic as a valuable tool for aiding in pathological diagnosis and for uncovering new insights into the mechanisms underlying transplant rejection.

How Much More Efficient Are Adaptive Platform Trials Than Multiple Stand-Alone Trials? A Comprehensive Simulation Study for Streamlining Drug Development During a Pandemic.

With the coronavirus disease 2019 (COVID-19) pandemic, there is growing interest in utilizing adaptive platform clinical trials (APTs), in which multiple drugs are compared with a single common control group, such as a placebo or standard-of-care group. APTs evaluate several drugs for one disease and accept additions or exclusions of drugs as the trials progress; however, little is known about the efficiency of APTs over multiple stand-alone trials. In this study, we simulated the total development period, total sample size, and statistical operating characteristics of APTs and multiple stand-alone trials in drug development settings for hospitalized patients with COVID-19. Simulation studies using selected scenarios reconfirmed several findings regarding the efficiency of APTs. The APTs without staggered addition of drugs showed a shorter total development period than stand-alone trials, but the difference rapidly diminished if patient's enrollment was accelerated during the trials owing to the spread of infection. APTs with staggered addition of drugs still have the possibility of reducing the total development period compared with multiple stand-alone trials in some cases. Our study demonstrated that APTs could improve efficiency relative to multiple stand-alone trials regarding the total development period and total sample size without undermining statistical validity; however, this improvement varies depending on the speed of patient enrollment, sample size, presence/absence of family-wise error rate adjustment, allocation ratio between drug and placebo groups, and interval of staggered addition of drugs. Given the complexity of planning and implementing APT, the decision to implement APT during a pandemic must be made carefully.

Clinical trial experience in Japan and future issues in developing drugs to treat COVID-19.

The National Center for Global Health and Medicine plays a central role in the treatment and research of infectious diseases in Japan. It has conducted various research and development activities on drugs to treat coronavirus disease 2019 (COVID-19) with clinical questions as starting points. Clinical trials are essential in developing new treatment modalities, but we have noticed some characteristic difficulties in clinical trials on emerging and re-emerging infectious diseases. For example, since there is no standard of care when an emerging infectious disease starts to spread, establishing an appropriate control group is complicated, and many things are hurried at the start of trials. This means there is little time to arrange a placebo, and conducting blinded, randomized, controlled trials has been difficult. Another issue characteristic of infectious disease has been that progress in enrolling subjects is affected by the spread of the disease. It was also a struggle to select institutions that provide medical care on the front lines of infectious disease and conduct clinical trials regularly. To start multicenter clinical trials expeditiously, a regulated and structured network is thus considered necessary. From the perspective of implementation, it is preferable to conduct decentralized clinical trials (DCTs) that do not depend on people coming to the medical institution, while from the perspective of preventing infections during the spread of COVID-19, wide adoption of eConsent is desirable. Based on the experience of COVID-19, new measures must be taken to prepare for emerging and re-emerging infectious diseases in the future.

Efficacy and Safety of Pre-Exposure Prophylaxis to Control HIV and Sexually Transmitted Infection Among Men Who Have Sex With Men: Protocol for a Single-Arm Interventional Study.

BACKGROUND: Pre-exposure prophylaxis (PrEP) against HIV infection is a new approach that involves the prophylactic use of the anti-HIV drug Truvada (tenofovir disoproxil fumarate [TDF] and emtricitabine [FTC]) by people not infected with HIV. OBJECTIVE: The objective of this investigator-initiated clinical study of PrEP was to evaluate the incidence of HIV and sexually transmitted infection (STI), safety and efficacy of PrEP in PrEP users, and their compliance with PrEP medication. The social, medical, and economic benefits of PrEP in Japan was assessed. METHODS: This single-center feasibility study of PrEP was conducted at the National Center for Global Health and Medicine, Tokyo, Japan, where a cohort of men who have sex with men without HIV was established in January 2017. This single-arm interventional study compared the efficacy and safety of PrEP in a single group of men who have sex with men who participated in PrEP cohort studies. For reference, the cohort study participants who did not participate in the PrEP study were included for comparison. Blood samples were collected for storage at baseline and clinic visits at 1, 3, and 6 months after starting PrEP and every 3 months thereafter. The participants were administered with 1 tablet of Truvada once daily as PrEP. They underwent blood and anal swab tests 1 and 3 months after starting PrEP and then HIV and STI infection assessments at 3-month intervals. Blood samples were centrifuged at the AIDS Clinical Center Laboratory. PrEP safety was evaluated by monitoring serum creatinine levels for symptoms of renal function disorders. The primary end point was the incidence of HIV in PrEP users (100 person-years). The secondary end points were the incidence of STI in PrEP users (100 person-years), incidence of adverse events caused by Truvada, frequency of high-risk sexual activity, and adherence to periodic visits and medication. RESULTS: The study protocol was reviewed and approved by the certified review board of the National Center for Global Health and Medicine (NCGM-C-003129-03) on April 20, 2020. Changes to the study plan were submitted for review by the certified review board and approved before implementation. Recruitment was completed on March 28, 2019, and the study was completed (last adult participant and last time point) on March 31, 2021. The data were analyzed, and the main results of the study have been published in a peer-reviewed journal. CONCLUSIONS: The findings indicated that PrEP is a highly effective and feasible strategy against HIV infection in terms of prophylactic response, retention, and compliance. TRIAL REGISTRATION: UMIN Clinical Trials Registry UMIN000031040; https://tinyurl.com/3msdkeb8 and Japan Registry of Clinical Trials jRCTs031180134; https://tinyurl.com/2p88mhyr. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR1-10.2196/50919.

Novel Complement C5 Small-interfering RNA Lipid Nanoparticle Prolongs Graft Survival in a Hypersensitized Rat Kidney Transplant Model.

BACKGROUND: Prophylaxis of antibody-mediated rejection (AMR) caused by donor-specific antibodies remains challenging. Given the critical roles of complement activity in antibody-mediated graft injury, we developed a lipid nanoparticle (LNP) formulation of small-interfering RNA against complement C5 (C5 siRNA-LNP) and investigated whether C5 siRNA-LNP could downregulate the complement activity and act as an effective treatment for AMR. METHODS: Lewis recipient rats were sensitized by skin grafting from Brown Norway donor rats. Kidney transplantation was performed at 4 wk post-skin grafting.C5 siRNA- or control siRNA-LNP was administered intravenously, and the weekly injections were continued until the study's conclusion. Cyclosporin (CsA) and/or deoxyspergualin (DSG) were used as adjunctive immunosuppressants. Complement activity was evaluated using hemolysis assays. The deposition of C5b9 in the grafts was evaluated using immunohistochemical analysis on day 7 posttransplantation. RESULTS: C5 siRNA-LNP completely suppressed C5 expression and complement activity (hemolytic activity ≤ 20%) 7 d postadministration. C5 siRNA-LNP in combination with CsA and DSG (median survival time: 56.0 d) prolonged graft survival compared with control siRNA-LNP in combination with CsA and DSG (median survival time: 21.0 d; P = 0.0012; log-rank test). Immunohistochemical analysis of the grafts revealed that downregulation of C5 expression was associated with a reduction in C5b9-positive area ( P = 0.0141, Steel-Dwass test). CONCLUSIONS: C5 siRNA-LNP combined with immunosuppressants CsA and DSG downregulated C5 activity and significantly prolonged graft survival compared with control siRNA-LNP with CsA and DSG. Downregulation of C5 expression using C5 siRNA-LNP may be an effective therapeutic approach for AMR.

NOD2 ligation subverts IFN-alpha production by liver plasmacytoid dendritic cells and inhibits their T cell allostimulatory activity via B7-H1 up-regulation.

The nucleotide-binding oligomerization domain (NOD)2/CARD15 protein, which senses muramyl dipeptide (MDP), a product of bacterial peptidoglycan, appears to play an important role in regulating intestinal immunity. Although the liver is exposed to gut-derived MDP, the influence of NOD2 ligation on hepatic APC, in particular dendritic cells (DC), is unknown. Freshly isolated mouse liver and spleen plasmacytoid (p)DC expressed higher levels of NOD2 message than conventional myeloid (m)DC. Following MDP stimulation in vivo, liver pDC, but not mDC, up-regulated expression of IFN regulatory factor 4 (IRF-4), a negative regulator of TLR signaling, and induced less allogeneic T cell proliferation and IFN-gamma production. The adoptive transfer of liver pDC from MDP-treated mice failed to prime allogeneic T cells in vivo. By contrast, splenic DC IRF-4 levels and T cell stimulatory activity remained unchanged. Liver pDC from MDP-stimulated mice also displayed greater IkappaBalpha, cell surface B7-H1, and B7-H1 relative to CD86 than control liver pDC. No similar effects were observed for liver mDC or spleen DC. Absence of B7-H1 on liver pDC reversed the inhibitory effect of MDP. After ex vivo stimulation with LPS or CpG, liver pDC but not mDC from MDP-treated animals secreted less IL-12p70, IL-6, and TNF-alpha and induced weaker allogeneic T cell proliferation than those from controls. Moreover, CpG-stimulated liver pDC from MDP-treated mice secreted less IFN-alpha than their splenic counterparts, and systemic levels of IFN-alpha were reduced in MDP-treated animals after CpG administration. These findings suggest that differential effects of NOD2 ligation on liver pDC may play a role in regulating hepatic innate and adaptive immunity.

Comparative evaluation of mechanical properties and shaping performance of heat-treated nickel titanium rotary instruments used in the single-length technique.

This study aimed to evaluate the mechanical properties of contemporary heat-treated nickel-titanium (NiTi) rotary instruments used in the single-length technique [ProTaper Next (PTN), HyFlex EDM (EDM), and JIZAI (JZ)]. Bending loads, cyclic fatigue resistance, torque/force values and canal-centering ratios were evaluated for the three instruments and a non-heat-treated experimental NiTi instrument with the same geometry as JZ (nJZ). EDM and JZ exhibited significantly lower bending load and more cycles to failure compared with nJZ and PTN (p<0.05). PTN and JZ exhibited significantly better centering ability than nJZ and EDM (p<0.05). JZ and nJZ generated significantly smaller upward force and maximum torque than PTN and EDM (p<0.05). Under the present experimental condition, JZ exhibited flexibility and cyclic fatigue resistance comparable to EDM, better maintained the canal curvature than the other instruments, and generated smaller torque and screw-in force than PTN and EDM.

Effect of Optimum Torque Reverse Motion on Torque and Force Generation during Root Canal Instrumentation with Crown-down and Single-length Techniques.

INTRODUCTION: Optimum torque reverse (OTR) motion is a torque-sensitive reciprocal motion in which the motor rotates in alternating 90° counterclockwise and 180° clockwise rotation when the torque exceeds a predetermined value. This study aimed to examine whether OTR motion contributes to torque and force reduction during nickel-titanium rotary instrumentation with the crown-down or single-length technique. METHODS: Twenty-eight simulated straight canals in resin blocks were divided into 2 groups according to the type of motion (OTR or continuous rotation). The groups were further subdivided according to the preparation technique (crown-down or single-length technique, n = 7 each). Automated root canal instrumentation was performed with a torque/force analyzing device (300 rpm, up-and-down speed of 10 mm/min) and EndoWave instruments (FKG Dentaire, La-Chaux-de-Fonds, Switzerland) to size #25/0.06 taper. Maximum torque and apical force were recorded and analyzed with analysis of variance and the Bonferroni test. RESULTS: During the crown-down preparation phase (#35/0.08, #30/0.06, #25/0.06, and #20/0.06), OTR motion developed lower maximum torque and upward force (representing the screw-in force) than continuous rotation. During the apical preparation phase (#25/0.06), OTR motion generated significantly lower maximum clockwise and counterclockwise torque (P < .05) when the single-length technique was used and significantly lower maximum upward force regardless of the preparation technique (P < .05) compared with continuous rotation. CONCLUSIONS: Under the present experimental conditions, OTR motion reduced both torque and screw-in force during the crown-down preparation phase of the crown-down technique and during the apical preparation phase of the single-length technique.

Enhanced root canal-centering ability and reduced screw-in force generation of reciprocating nickel-titanium instruments with a post-machining thermal treatment.

This study aimed to evaluate the influence of a post-machining thermal treatment on canal-centering ability and torque/force generation of reciprocating nickel-titanium instruments. Simulated J-shaped resin canals were prepared with reciprocating instruments sharing identical geometric architecture and with/without post-machining thermal treatment (Reciproc Blue/Reciproc, VDW, Munich, Germany). Using an original automated root canal instrumentation and torque/force analyzing device, files were operated in a combination of reciprocation and up-and-down motion, and torque/force values were monitored. Canal-centering ratios were measured after superimposition of pre- and post-instrumentation images. Compared with Reciproc, Reciproc Blue showed a significantly lower canal-centering ratio (i.e., less deviation; p<0.05) at 0-1 mm from the apex and generated a significantly smaller upward maximum vertical force (p<0.05). Under standardized conditions using the automated device, Reciproc Blue showed better canal-centering ability and reduced screw-in forces than Reciproc, indicating that the post-machining thermal treatment confers superior performance to reciprocating nickel-titanium instruments.

Inhibition of Kupffer cell-mediated early proinflammatory response with carbon monoxide in transplant-induced hepatic ischemia/reperfusion injury in rats.

UNLABELLED: Proinflammatory responses play critical roles in hepatic ischemia/reperfusion (I/R) injury associating with liver transplantation (LTx), and carbon monoxide (CO) can effectively down-regulate them. Using wild-type (WT) to enhanced green fluorescent protein (EGFP)-transgenic rat LTx with 18-hour cold preservation in University of Wisconsin solution, this study analyzed the relative contribution of donor and host cells during early posttransplantation period and elucidated the mechanism of hepatic protection by CO. CO inhibited hepatic I/R injury and reduced peak alanine aminotransferase levels at 24 hours and hepatic necrosis at 48 hours. Abundant EGFP(+) host cells were found in untreated WT liver grafts at 1 hour and included nucleated CD45(+) leukocytes (myeloid, T, B, and natural killer cells) and EGFP(+) platelet-like depositions in the sinusoids. However, reverse transcription polymerase chain reaction (RT-PCR) analysis of isolated graft nonparenchymal cells (NPCs) revealed that I/R injury-induced proinflammatory mediators [for example, tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS)] were not up-regulated in purified CD45(+) cells of donor or host origin. Instead, TNF-alpha and IL-6 messenger RNA (mRNA) elevation was exclusively seen in isolated CD68(+) cells, whereas iNOS mRNA up-regulation was seen in hepatocytes. Nearly all CD68(+) cells at 1 hour after LTx were EGFP(-) donor Kupffer cells, and CO efficiently inhibited TNF-alpha and IL-6 up-regulation in the CD68(+) Kupffer cell fraction. When graft Kupffer cells were inactivated with gadolinium chloride, activation of inflammatory mediators in liver grafts was significantly inhibited. Furthermore, in vitro rat primary Kupffer cell culture also showed significant down-regulation of lipopolysaccharide (LPS)-induced inflammatory responses by CO. CONCLUSION: These results indicate that CO ameliorates hepatic I/R injury by down-regulating graft Kupffer cells in early postreperfusion period. The study also suggests that different cell populations play diverse roles by up-regulating distinctive sets of mediators in the acute phase of hepatic I/R injury.

Assessment of mechanical properties of WaveOne Gold Primary reciprocating instruments.

This study aimed to evaluate cyclic fatigue resistance and bending properties and torque/force generation of WaveOne Gold (WOG) Primary in comparison with WaveOne (WO) Primary and Reciproc (RE) R25. A cyclic fatigue test revealed that the WOG Primary took significantly longer time to fracture compared with the WO Primary (p<0.05). The WOG Primary had the smallest load values at a deflection of 0.5 and 2 mm (p<0.05), as measured with a cantilever bending test. Torque/force measurement demonstrated that maximum upward force and maximum counterclockwise torque values in the WOG Primary were significantly lower than those in the RE R25 (p<0.05). Under the present experimental condition, the WOG Primary showed a higher cyclic fatigue resistance compared with the WO Primary, a higher flexibility compared with the WO Primary and RE R25, and generated a significantly lower maximum torque compared with the RE R25.

Effect of Different Speeds of Up-and-down Motion on Canal Centering Ability and Vertical Force and Torque Generation of Nickel-titanium Rotary Instruments.

INTRODUCTION: This study was performed to evaluate how the speed of up-and-down motion affects the canal centering ability and torque/force generation of ProTaper Next rotary instruments (Dentsply Maillefer, Ballaigues, Switzerland). METHODS: Twenty-one simulated resin canal blocks with a J-shaped canal were prepared with ProTaper Next X1, X2, and X3 instruments using an original automated root canal instrumentation and torque/force analyzing device with up-and-down speed settings of 10, 50, and 100 mm/min (low-, medium-, and high-speed groups, respectively). Pre- and postinstrumentation images were superimposed, and centering ratios were calculated at 0-3 mm from the apex. The maximum vertical force and torque were also recorded. The results were statistically analyzed using 1-way analysis of variance and the Tukey test. RESULTS: At 0, 0.5, 1, and 2 mm from the apex, the high-speed group showed the lowest centering ratio (ie, least deviation) followed by the medium-speed and low-speed groups (P < .05). Force values (downward and upward) tended to increase as the up-and-down speed increased; with the X2 and X3 instruments, the high-speed and/or medium-speed groups generated significantly larger values than the low-speed group (P < .05). With all instruments, the high-speed and/or medium-speed groups generated significantly larger clockwise torque than the low-speed group (P < .05). One and 2 X2 instruments fractured in the low- and high-speed groups, respectively. CONCLUSIONS: The up-and-down speed affected the canal centering ability and stress generation of ProTaper Next instruments. The high-speed group showed the best centering ability but tended to generate larger vertical force and torque than the medium- and low-speed groups.

High PD-L1/CD86 ratio on plasmacytoid dendritic cells correlates with elevated T-regulatory cells in liver transplant tolerance.

BACKGROUND: Both dendritic cells (DC) and T-regulatory cells (Treg) have been implicated in regulation of alloimmune responses and transplant tolerance. METHODS: We analyzed B7 coregulatory molecule expression on circulating DC subset precursors, together with CD4+CD25(hi) Foxp3+ Treg by rare event, flow cytometric analysis in operationally tolerant pediatric liver transplant recipients (TOL), those undergoing prospective immunosuppressive drug weaning (PW) or maintenance immunosuppression (MI), and normal healthy individuals (controls). RESULTS: Use of DC subset-specific monoclonal antibodies confirmed elevated precursor plasmacytoid DC/myeloid DC ratios in TOL and PW compared with MI. In addition, Treg frequencies were higher in TOL than in PW and MI, but not controls. While there was no difference in levels of costimulatory and coinhibitory molecules on precursor myeloid DC between the groups, the programmed death ligand-1 (PD-L1=B7-H1):CD86 (B7-2) ratio on precursor plasmacytoid DC was significantly higher in TOL than MI and correlated with the Treg frequency. There was no relation between prednisone or tacrolimus dose or tacrolimus trough level and either the PD-L1/CD86 ratio on plasmacytoid DC or the Treg frequency. Moreover, clinically relevant concentrations of dexamethasone or tacrolimus did not affect these values in short-term culture. CONCLUSION: These novel findings suggest a possible functional relationship between the enhanced incidence of precursor plasmacytoid DC, their comparatively high relative expression of the coinhibitory molecule PD-L1, and the elevated frequency of Treg in operational liver transplant tolerance.

Dendritic cells, the liver, and transplantation.

Interstitial liver dendritic cells (DCs) exhibit phenotypic diversity and functional plasticity. They play important roles in both innate and adaptive immunity. Their comparatively low inherent T cell stimulatory ability and the outcome of their interactions with CD4(+) and CD8(+) T cells, as well as with natural killer (NK) T cells and NK cells within the liver, may contribute to regulation of hepatic inflammatory responses and liver allograft outcome. Liver DCs migrate in the steady state and after liver transplantation to secondary lymphoid tissues, where the outcome of their interaction with antigen-specific T cells determines the balance between tolerance and immunity. Systemic and local environmental factors that are modulated by ischemia-reperfusion injury, liver regeneration, microbial infection, and malignancy influence hepatic DC migration, maturation, and function. Current research in DC biology is providing new insights into the role of these important antigen-presenting cells in the complex events that affect liver transplant outcome.

Sphingosine 1-phosphate receptor agonism impairs skin dendritic cell migration and homing to secondary lymphoid tissue: association with prolonged allograft survival.

The novel immunomodulator FTY720 is a prototypic sphingosine-1-phosphate (S1P) receptor agonist that regulates lymphocyte migration and prolongs allograft survival. Skin dendritic cells (DC) play important roles in cutaneous immunity. We investigated the migration and function of skin DC exposed to FTY720 in vivo, or to its metabolite FTY720 phosphate (P) in vitro. C57BL/10 (H2(b)) recipient (but not donor) FTY720 treatment prolonged median skin C3H (H2(k)) allograft survival significantly, from 12 to 34.5 days. Non-transplanted, FTY720-treated mice revealed a marked increase in skin DC, although total DC in skin-draining lymph nodes (DLN) were unchanged compared with controls. Fewer allogeneic donor DC migrated to DLN of FTY720-treated graft recipients. DC that migrated from the skin of FTY720-treated mice showed reduced MHC class II, CD86 and CCR7 expression, suggesting impaired migratory potential to secondary lymphoid tissue, that correlated with DC retention in skin, and reduced T cell stimulatory activity. Fewer DC migrated from normal skin explants exposed to the FTY720 metabolite, FTY720P than to control medium. DC that did migrate expressed lower levels of MHC class II, CD86 and CCR7, and inferior T cell stimulatory ability. These data demonstrate S1P signaling regulates skin DC trafficking and modulates MHC class II, costimulatory, and homing receptor molecule expression that impairs their ability to elicit allogeneic T cell responses.

Comparative analysis of mechanical properties of differently tapered nickeltitanium endodontic rotary instruments.

This study aimed to compare the mechanical properties of differently-tapered EndoWave nickel-titanium endodontic rotary instruments (size #30/0.04 taper and #30/0.06 taper; Group 0.04 and 0.06, respectively). Torsional and bending properties were evaluated with the cyclic fatigue test and the cantilever bending test, respectively. Canal transportation was assessed following instrumentation of curved canals in resin blocks. Torque and apical force during instrumentation were determined using an automated instrumentation and torque/force analyzing device. The number of cycles to fracture was Group 0.04>Group 0.06 (p<0.05). The bending load values were Group 0.06>Group 0.04 (p<0.05). Group 0.04 better maintained the original canal shape compared with Group 0.06. The median clockwise torque value was Group 0.06>Group 0.04 (p<0.05), whereas no significant differences were detected in the median counterclockwise torque and apical force values (p>0.05). Under the present experimental condition, 0.04- and 0.06-tapered EndoWave instruments exhibited different mechanical properties.

Evaluation of selected mechanical properties of NiTi rotary glide path files manufactured from controlled memory wires.

This study aimed to investigate mechanical properties related to flexibility and fracture resistance of controlled memory wiremanufactured nickel-titanium rotary glide path files [HyFlex EDM Glide Path File (EDM) and HyFlex GPF (GPF)]. Scout RaCe (RaCe) served as control. Bending loads, torsional/cyclic fatigue resistance, and screw-in forces were measured. EDM showed a significantly larger torque at fracture, a longer time to cyclic fracture in reciprocation and a larger screw-in force compared with GPF and RaCe. GPF showed significantly lower bending loads and higher angular deflection values than EDM and RaCe, and a significantly longer time to cyclic fracture than RaCe. The time to cyclic fracture was significantly longer in reciprocation compared with continuous rotation in EDM and GPF. It can be concluded that EDM and/or GPF showed higher flexibility and cyclic/torsional fatigue resistance compared with RaCe; and that reciprocation conferred better cyclic fatigue resistance to EDM and GPF.

Infusion of stably immature monocyte-derived dendritic cells plus CTLA4Ig modulates alloimmune reactivity in rhesus macaques.

BACKGROUND: Immature dendritic cells (DC) can promote long-term transplant survival in rodents. We assessed the impact of stably immature, donor-derived DC on alloimmune reactivity in rhesus macaques. METHODS: CD14 monocytes isolated from leukapheresis products of Macacca mulatta were cultured in granulocyte-macrophage colony stimulating factor plus interleukin (IL)-4+/-vitamin (vit) D3, and IL-10. Major histocompatibility complex class II and cosignaling molecule expression was determined on CD11c cells by flow cytometry. T-cell allostimulatory capacity of the DC, including DC exposed to proinflammatory cytokines, was determined in mixed leukocyte reaction. To test their influence in vivo, purified DC were infused intravenously into allogeneic recipients, either alone or followed by CTLA4Ig, 24 hr later. Proliferative responses of recipient CFSE-labeled T cells to donor or third party DC, cytokine production by stimulated T cells, and circulating alloantibody levels were determined by flow cytometry, up to 100 days postinfusion. RESULTS: VitD3/IL-10-conditioned, monocyte-derived DC were resistant to maturation and failed to induce allogeneic T cell proliferation in vitro. After their infusion, an increase in anti-donor and anti-third party T-cell reactivity was observed, that subsequently subsided to fall significantly below pretreatment levels (by day 56) only in animals also given CTLA4Ig. No increase in circulating immunoglobulin (Ig) M or IgG anti-donor alloantibody titers compared with pretreatment values was detected. With DC+CTLA4Ig infusion, alloreactive IL-10-producing T cells were prevalent in the circulation after day 28. CONCLUSIONS: Maturation-resistant rhesus DC infusion is well-tolerated. DC+CTLA4Ig infusion modulates allogeneic T-cell responses and results in hyporesponsiveness to donor and third party alloantigens.