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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.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.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).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.

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

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.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.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.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.

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

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

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 0.04Group 0.06 (p0.05). The bending load values were Group 0.06Group 0.04 (p0.05). Group 0.04 better maintained the original canal shape compared with Group 0.06. The median clockwise torque value was Group 0.06Group 0.04 (p0.05), whereas no significant differences were detected in the median counterclockwise torque and apical force values (p0.05). Under the present experimental condition, 0.04- and 0.06-tapered EndoWave instruments exhibited different mechanical properties.

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.

The purpose of the present study was to compare 2 modes of reciprocal movement (torque-sensitive and time-dependent reciprocal rotation) with continuous rotation in terms of torque and apical force generation during nickel-titanium rotary root canal instrumentation.A custom-made automated root canal instrumentation and torque/force analyzing device was used to prepare simulated canals in resin blocks and monitor the torque and apical force generated in the blocks during preparation. Experimental groups (n = 7, each) consisted of (1) torque-sensitive reciprocal rotation with torque-sensitive vertical movement (group TqR), (2) time-dependent reciprocal rotation with time-dependent vertical movement (group TmR), and (3) continuous rotation with time-dependent vertical movement (group CR). The canals were instrumented with TF Adaptive SM1 and SM2 rotary files (SybronEndo, Orange, CA), and the torque and apical force were measured during instrumentation with SM2. The mean and maximum torque and apical force values were statistically analyzed using 1-way analysis of variance and the Tukey test (α = 0.05).The recordings showed intermittent increases of upward apical force and clockwise torque, indicating the generation and release of screw-in forces. The maximum upward apical force values in group TmR were significantly smaller than those in group CR (P .05). The maximum torque values in clockwise and counterclockwise directions in groups TqR and TmR were significantly smaller than those in group CR (P .05).Under the present experimental conditions using TF Adaptive instruments, both torque-sensitive and time-dependent reciprocal rotation generated significantly lower maximum torque and may have advantages in reducing stress generation caused by screw-in forces when compared with continuous rotation.

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

This study examined the dynamic fracture behavior of nickel-titanium rotary instruments in torsional or cyclic loading at continuous or reciprocating rotation by means of high-speed digital video imaging.The ProFile instruments (size 30, 0.06 taper; Dentsply Maillefer, Ballaigues, Switzerland) were categorized into 4 groups (n = 7 in each group) as follows: torsional/continuous (TC), torsional/reciprocating (TR), cyclic/continuous (CC), and cyclic/reciprocating (CR). Torsional loading was performed by rotating the instruments by holding the tip with a vise. For cyclic loading, a custom-made device with a 38° curvature was used. Dynamic fracture behavior was observed with a high-speed camera. The time to fracture was recorded, and the fractured surface was examined with scanning electron microscopy.The TC group initially exhibited necking of the file followed by the development of an initial crack line. The TR group demonstrated opening and closing of a crack according to its rotation in the cutting and noncutting directions, respectively. The CC group separated without any detectable signs of deformation. In the CR group, initial crack formation was recognized in 5 of 7 samples. The reciprocating rotation exhibited a longer time to fracture in both torsional and cyclic fatigue testing (P .05). The scanning electron microscopic images showed a severely deformed surface in the TR group.The dynamic fracture behavior of NiTi rotary instruments, as visualized with high-speed digital video imaging, varied between the different modes of rotation and different fatigue testing. Reciprocating rotation induced a slower crack propagation and conferred higher fatigue resistance than continuous rotation in both torsional and cyclic loads.

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).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.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.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.

Donor–π–acceptor (D–π–A) type pyridinium dye bearing an iodide ion as the counter anion that has been newly synthesized showed specific solvatochromism, leading to large bathochromic shift of absorption band in halogenated solvents: the bathochromic shifts of the D–π–A type pyridinium dye in halogenated solvents are larger than those of the non-halogenated solvents of low ɛr values. Investigation of absorption spectral measurement, 1H NMR measurements, and semi-empirical molecular calculations (AM1 and INDO/S using the SCRF Onsager Model) revealed that the intramolecular charge transfer (ICT) characteristics of the D–π–A type pyridinium dye became stronger in the halogenated solvents. On the basis of the experimental results and the theoretical calculations, the influences of halogenated solvent on the large bathochromic shift of D–π–A type pyridinium dye are discussed. ©2012 Elsevier Science. All rights reserved.

Background To evaluate the role of the oral glucose tolerance test (OGTT) before transplantation and to examine the risk factors for new-onset diabetes after transplantation (NODAT) during long-term follow-up of renal transplant recipients receiving FK-based therapy. Methods The study evaluated 378 patients pre-transplantation using the OGTT and assigned them to one of three groups: Group 1, normal pattern; Group 2, impaired fasting glucose (IFG)/impaired glucose tolerance (IGT) pattern (IFG/IGT); and Group 3, DM pattern. Results Although the incidence of NODAT was higher in Group 3 than in groups 1 and 2, no significant difference was found between the three groups with regard to graft survival during long-term follow-up. Multivariate analysis showed that only a family history of diabetes was a significant factor determining NODAT progression. Conclusions Impaired glucose tolerance appears to be a threshold influencing NODAT; however, it was not a significant factor in graft survival. Careful monitoring and management based on the result of the pre-transplantation OGTT appear to prevent the deterioration of impaired glucose tolerance in renal transplant recipients receiving FK-based therapy, even when a pre-operative OGTT shows impaired glycemic control.

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-γ 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 IκBα, 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-α and induced weaker allogeneic T cell proliferation than those from controls. Moreover, CpG-stimulated liver pDC from MDP-treated mice secreted less IFN-α than their splenic counterparts, and systemic levels of IFN-α 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.

Bis(triethoxysilyl)-substituted oligothiophene with a moderate π-conjugation length was newly synthesized and polymerized to give a polysilsesquioxane network having oligothiophene units dispersed homogeneously without phase separation. The polymer was fixed on glass or ITO substrate by spin-coating and annealing. The resulting film exhibited a high mechanical strength due to the covalent bonding with the substrates, and was electrochemically stable even after 300 redox cycles in electrolyte solution. Chemical oxidation of the polymer films yielded electrically conductive and almost transparent films.

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.

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.

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.

For many years, cellular assays, including mixed leukocyte reactions, cell-mediated lympholysis, and T helper and T cytotoxic cell precursor frequency analyses, have been used to monitor the immune response status of organ allograft recipients. The predictive value, sensitivity, and specificity of such assays have often been questioned. In addition, no in vitro assay has been shown to correlate reliably with or to predict operational tolerance. As tolerogenic strategies are introduced into clinical testing, the need for development and validation of “tolerance” assays is great. Herein, we survey contemporary and emerging technologic approaches to confront this challenge.

A portal venous injection of allogeneic donor cells is known to prolong the survival of subsequently transplanted allografts. In this study, we investigated the role of liver sinusoidal endothelial cells (LSECs) in immunosuppressive effects induced by a portal injection of allogeneic cells on T cells with indirect allospecificity. To eliminate the direct CD4+ T cell response, C57BL/6 (B6) MHC class II-deficient C2tatm1Ccum (C2D) mice were used as donors. After portal injection of irradiated B6 C2D splenocytes into BALB/c mice, the host LSECs that endocytosed the irradiated allogeneic splenocytes showed enhanced expression of MHC class II molecules, CD80, and Fas ligand (FasL). Due to transmigration across the LSECs from BALB/c mice treated with a portal injection of B6 C2D splenocytes, the naive BALB/c CD4+ T cells lost their responsiveness to stimulus of BALB/c splenic APCs that endocytose donor-type B6 C2D alloantigens, while maintaining a normal response to stimulus of BALB/c splenic APCs that endocytose third-party C3H alloantigens. Similar results were not observed for naive BALB/c CD4+ T cells that transmigrated across the LSECs from BALB/c FasL-deficient mice treated with a portal injection of B6 C2D splenocytes. Adaptive transfer of BALB/c LSECs that had endocytosed B6 C2D splenocytes into BALB/c mice via the portal vein prolonged the survival of subsequently transplanted B6 C2D hearts; however, a similar effect was not observed for BALB/c FasL-deficient LSECs. These findings indicate that LSECs that had endocytosed allogeneic splenocytes have immunosuppressive effects on T cells with indirect allospecificity, at least partially via the Fas/FasL pathway.

DC are believed to play important roles in the induction and regulation of immune responses in the liver, an organ implicated in peripheral tolerance. Since the liver is located downstream of the gut, it is constantly exposed to bacterial LPS. Our recent observations indicate that prior exposure to endotoxin modulates subsequent liver DC responses to this TLR4 ligand. In this study, we demonstrate that endotoxin modifies the capacity of mouse liver myeloid DC (MDC) activated by CpG (TLR9 ligand) to direct Th1-type responses. IL-12 production by liver MDC was significantly lower than that of spleen MDC following CpG or Imiquimod (R837; TLR7 ligand) activation in vitro. In addition, allogeneic T cells stimulated by CpG-activated liver MDC secreted significantly lower levels of IFN-gamma than T cells stimulated with CpG-activated spleen MDC. A similar effect on liver DC was observed in response to in vivo CpG administration. This effect may be explained by exposure of the DC to endotoxin, because LPS attenuated IL-12 production by CpG-stimulated liver MDC, both in vitro and in vivo. Moreover, attenuation of the response to CpG was not observed in liver MDC from TLR4-mutant (C3H/HeJ) mice, in which TLR4 signaling is impaired. These data suggest that endotoxin-induced 'cross-tolerance' to TLR ligands in liver DC may contribute to down-regulation of hepatic immune responses.

More effective discrimination between CD4+CD25+ regulatory T cells (Treg) and activated T cells would significantly improve the current level of purification of Treg and their therapeutic application. We observed that ∼90% of Treg (positive for the nuclear transcription factor Forkhead winged helix protein-3 and able to inhibit naive T cell proliferation) isolated from the spleens or lymph nodes of normal mice did not express significant levels of the inhibitory receptor programmed cell death-1 (PD-1) on their surface, but retained PD-1 intracellularly. An identical phenotype was also identified for human CD4+CD25high T cells isolated from peripheral blood of healthy volunteers. By contrast, activated T cells expressed high levels of surface PD-1 that paralleled up-regulation of CD25 during effector cell expansion. This distinction allowed us to isolate CD4+CD25+PD-1− T cells with suppressive activity from mice immunized with mature allogeneic dendritic cells. Although purification was limited to resting Treg because TCR ligation induced up-regulation of surface PD-1, this strategy nevertheless represents a valuable step toward more definitive characterization of Treg and their improved purification for therapeutic assessment.

Although livers transplanted across MHC barriers in mice are normally accepted without recipient immune suppression, the underlying mechanisms remain to be clarified. To identify the cell type that contributes to induction of such a tolerance state, we established a mixed hepatic constituent cell-lymphocyte reaction (MHLR) assay. Irradiated C57BL/6 (B6) or BALB/c mouse hepatic constituent cells (HCs) and CFSE-labeled B6 splenocytes were cocultured. In allogeneic MHLR, whole HCs did not promote T cell proliferation. When liver sinusoidal endothelial cells (LSECs) were depleted from HC stimulators, allogeneic MHLR resulted in marked proliferation of reactive CD4+ and CD8+ T cells. To test the tolerizing capacity of the LSECs toward alloreactive T cells, B6 splenocytes that had transmigrated through monolayers of B6, BALB/c, or SJL/j LSECs were restimulated with irradiated BALB/c splenocytes. Nonresponsiveness of T cells that had transmigrated through allogeneic BALB/c LSECs and marked proliferation of T cells transmigrated through syngeneic B6 or third-party SJL/j LSECs were observed after the restimulation. Transmigration across the Fas ligand-deficient BALB/c LSECs failed to render CD4+ T cells tolerant. Thus, we demonstrate that Fas ligand expressed on naive LSECs can impart tolerogenic potential upon alloantigen recognition via the direct pathway. This presents a novel relevant mechanism of liver allograft tolerance. In conclusion, LSECs are capable of regulating a polyclonal population of T cells with direct allospecificity, and the Fas/Fas ligand pathway is involved in such LSEC-mediated T cell regulation.

We demonstrated that an indirect pathway of alloantigen presentation via liver sinusoidal endothelial cells (LSEC) is involved in alloreactive T-cell tolerance induced by portal venous injection (PI) of donor cells. Thirty million C57BL/6 (B6) splenocytes that were either untreated or treated with 30-Gy irradiation were injected via the portal vein into Balb/c mice. Host LSEC expressing major histocompatibility complex class II actively endocytosed the allogeneic naive splenocytes as well as irradiated splenocytes after PI. Using a transendothelial migration assay, it was demonstrated that host-type Balb/c CD4(+) T cells that transmigrated across LSEC that had captured irradiated B6 splenocytes were rendered tolerant to subsequent alloantigen presentation by host professional antigen-presenting cells. Consistently, PI of irradiated donor-type splenocytes led to remarkable prolongation of the survival of subsequently transplanted heart allografts. These results indicate that indirect antigen presentation by LSEC significantly contributes to alloreactive T-cell tolerance induced by PI of irradiated donor splenocytes.

Livers transplanted across major histocompatibility complex (MHC) barriers in mice are normally accepted without recipient immune suppression. To identify the cell type that contributes to induction of such a tolerance state, we established an allogeneic mixed hepatic constituent cell-lymphocyte reaction (MHLR) assay. Hepatic constituent cells were isolated from C57BL/6 (B6) and Balb/c mice as stimulators, and splenocytes were isolated from B6 mice as responders. Irradiated hepatic constituent cells were co-cultured with fluorescent dye (CFSE)-labeled B6 splenocytes. In the allogeneic MHLR using either whole hepatic constituent cells or parenchymal hepatocytes as stimulators, a lack of T-cell proliferation was observed. Only when CD105(+) cells, which are exclusively liver sinusoidal endothelial cells (LSECs), were depleted from hepatic constituent cell stimulators, the MHLR resulted in marked proliferation of both allo-reactive CD4(+) and CD8(+) T cells. These results indicate that CD105(+) LSECs have the capacity to induce nonresponsiveness of T cells across MHC barriers.

Although it is known that activation of natural killer (NK) cells causes liver injury, the mechanisms underlying NK cell-induced killing of self-hepatocytes are not clear. We demonstrated that liver NK cells have cytotoxicity against normal syngeneic hepatocytes in mice. Polyinosinic-polycytidylic acid (poly I:C) treatment enhanced hepatocyte toxicity of liver NK cells but not that of spleen NK cells. Unlike NK cells in other tissues, approximately 30%-40% of liver NK cells constitutively express tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). An in vitro NK cell cytotoxic assay revealed that hepatocyte toxicity of liver NK cells from both naïve and poly I:C-treated mice was inhibited partially by an anti-TRAIL monoclonal antibody (mAb) alone and completely by the combination with anti-Fas ligand (FasL) mAb and a perforin inhibitor, concanamycin A, indicating contribution of TRAIL to NK cell-mediated hepatocyte toxicity. The majority of TRAIL(+) NK cells lacked expression of Ly-49 inhibitory receptors recognizing self-major histocompatibility complex class I, indicating a propensity to targeting self-hepatocytes. Poly I:C treatment significantly upregulated the expression of Ly-49 receptors on TRAIL(-) NK cells. This might be a compensatory mechanism to protect self-class I-expressing cells from activated NK cell-mediated killing. However, such compensatory alteration was not seen at all in the TRAIL(+) NK cell fraction. Thus, liver TRAIL(+) NK cells have less capacity for self-recognition, and this might be involved in NK cell-dependent self-hepatocyte toxicity. In conclusion, our findings are consistent with a model in which TRAIL-expressing NK cells play a critical role in self-hepatocyte killing through poor recognition of MHC.

Human antibodies (Abs) against blood group A or B carbohydrate determinant are a major barrier to ABO-incompatible organ transplantation; however, the phenotype and other properties of B cell types responding to A or B carbohydrate epitopes have not been defined. Studies here, which use fluorescein-labeled synthetic A determinant (GalNAcalpha1-3Fucalpha1-2Gal), demonstrate that B cells bearing surface IgM (sIgM) receptors recognizing blood group A carbohydrate determinant are found exclusively in a small B cell subpopulation, i.e. sIgM+ CD11b+ CD5+ B1 cells, in blood group O human peripheral blood mononuclear cells (PBMC). In order to test anti-A Abs producing capacity of the human PBMC, nonobese diabetic (NOD)/severe combined immune-deficient (SCID) mice that have been treated with rabbit anti-asialo GM1 serum to deplete natural killer cells and with 3 Gy of whole body irradiation were engrafted with blood group O or A human PBMC, followed by sensitization of human blood group A red blood cells. Anti-A-specific human Abs were detected in the sera of the mice that received blood group O human PBMC, whereas they were not detected in the sera of the mice that received blood group A human PBMC, indicating profound tolerance of auto-reactive B cells. The human PBMC-NOD/SCID chimera developed by injection of blood group O human PBMC might be a useful in vivo model to test effects of immunosuppressants or other approaches on human B cells that respond to blood group A antigens.

Background. This study was performed to determine the usefulness of intraoperative near-infrared spectroscopy (NIRS) for evaluating the extent of congestion in the anterior segment of the graft after living-donor liver transplantation using right lobe grafts that do not have the middle hepatic vein.

Poor bone marrow (BM) engraftment in a xenogeneic combination results at least in part from the limited engraftment capacity of BM-derived stromal cells, which support hematopoietic repopulation in a species-specific fashion. We attempted to construct a BM stromal microenvironment by engraftment of BM plug fragments into kidney capsules in a rat-to-mouse combination. BM plugs from F344/N Jcl-rnu/rnu (F344 nu) rats were transplanted into the kidney capsules of C.B-17 scid/scid (C.B-17 scid) mice treated with rabbit anti-asialo-GM1 serum to deplete natural killer (NK) cells and then with 3 Gy of whole body irradiation. As a conventional control, an equivalent amount of F344 nu bone marrow cells (BMCs) was intravenously injected into C.B-17 scid mice treated with a similar conditioning regimen. In both mouse recipients of rat BM plug engraftment in the kidney capsules and recipients of intravenous injection of rat BMC suspension, comparable extents of donor rat class I+ cells were persistently detected in the peripheral blood. However, the differentiation of rat-derived B cells in the mouse recipients of rat BM plugs was more rapid than that in the recipients of rat BMC suspension. In the late phase (10 weeks after BM transplantation), the percentage of rat-derived T cells (CD4+ cells) in the mouse recipients of rat BM plugs was significantly higher than that in the recipients of rat BMC suspension. At this time point, ectopic BM structure consisting of bone, mesenchymal cells, and hematopoietic progenitors was constructed in the kidney capsules of mice that received rat BM plugs. Most of the cells in the ectopic BM were derived from the donor rat. Thus, engraftment of BM plugs into the kidney capsules results in the construction of a donor-derived BM microenvironment, facilitating multilineage mixed xenogeneic chimerism.

当センターでこの12年間に経験したpositive selectionのCAPD症例59例中, 死亡15例, 血液透析 (HD) への移行23例の計38例 (64.4%) が, CAPD脱落症例であった.これらの脱落原因の50%がCAPDに関連したもので, 中でもHD移行の主な理由は, 限外濾過不全, 難治性・頻回のCAPD腹膜炎, 硬化性被嚢性腹膜炎 (SEP) であった. また, 開腹手術を要した6例中5例がHDへ移行した. 脱落関連因子として, CAPD施行期間の長さ, CAPD腹膜炎罹患が有意に相関があると考えられた.SEP症例は, 59例中3例, 5.1%であった. 限外濾過不全のためHD移行後, この前段階と考えられる著明な腹水貯留4症例に, 発症早期からのステロイド治療が有効であった.CAPD排液中のamylase値は, 平常時およびCAPD腹膜炎時には, それぞれ, 5.6±2.7, 19.3±22.6mU/mlで, 後者で有意に高値を呈した. 穿孔性腹膜炎2例 (S状結腸, 回腸) と急性膵炎1例に緊急開腹手術を施行したが, 術前のこれらの値はさらに著明な高値を示し, 一般のCAPD腹膜炎との鑑別に極めて有用であった. 回腸穿孔例では, 術後に縫合不全を合併し手術死亡となったが, このような後期の腹膜硬化症を呈する症例においては, 腸管吻合に重大な危険性が存在すると考えられた.脱落症例38例中16例に, カテーテル抜去時や開腹手術時に腹膜のサンプリングが行われ, 病理組織学的検索がなされた. CAPD施行期間が8-18か月の3例では, いずれも腹膜線維症の所見であった. しかし, 60か月以上の13例中12例, 92.3%では, 腹膜機能障害が非可逆性といわれる中期以後の腹膜硬化症の所見 (中期5例, 後期7例) が認められ, 極めて注目すべき結果が得られた. この事実は, かかる長期CAPD症例においては, SEPとの関連から, その中止時期も含めて, 注意深い経過観察が必要であると考えられた.

症例は60歳, 女性. 1986年12月9日慢性糸球体腎炎による慢性腎不全と診断され, 持続腹膜透析 (CAPD) 導入. その直後の1987年2月12日両側手根管症候群にて開放術施行. 以後9年間で腹膜炎を2回発症. 徐々に除水能の低下をきたし, 1995年12月より血液濾過透析に移行. 透析低血圧および, 凝固能異常の合併によると思われる内シャントの閉塞を反復した. 1996年11月より悪心, 嘔吐, 腹部膨満等の腸閉塞症状出現, 保存的療法にて-時軽快. 消化管透視腹部超音波検査, 腹部CT検査で, 腸管の拡張, 腸蠕動の著明な低下が認められ, 硬化性被嚢性腹膜炎と診断された. 同年12月再び腸閉塞出現, 保存的加療施行するも症状遷延. 内シャント再建術中, 突然ショック状態を呈し, 急性心不全状態持続したのち, 重篤な心筋伝導障害で12月16日死亡した. 病理剖検所見では, 化膿性腹膜炎および腹膜の線維性硬化像に加えて, 全身骨・関節, 消化管, 心, 肺, その他諸臓器にアミロイド沈着を認め, 硬化性被嚢性腹膜炎およびアミロイドーシスの合併が判明した. 本例のアミロイドは, β2-microglobulin由来であったが, AA抗体陽性, 過マンガン酸カリ抵抗性で, 従来の文献とは異なっていた.透析に伴う重篤な合併症を重複した, 貴重な剖検例を経験した. 透析療法の進歩で, 慢性腎不全患者の長期生存が可能となった現在, 同様の症例の出現が今後も予想され, 本例を長期透析患者の全身管理の指針について, 貴重な布石としたい.

症例は63歳の男性で, 主訴は下血. 血清CA19-9異常高値 (830U/ml), CT, Gaシンチなどから, 悪性腸間膜腫瘍を疑い開腹した. 回盲部より80cmの部位にMeckel憩室を認め, 同部に腫瘍および腸間膜リンパ節の腫脹を認めたため, リンパ節郭清を含めた回腸切除を行った. 病理組織学的にはMeckel憩室から発生した中分化型腺癌で, 憩室内には迷入組織を認めなかった. 術後血清CA19-9は速やかに低下し, また抗CA19-9抗体を用いた免疫染色で陽性であり, CA19-9産生腫瘍と診断した. Meckel憩室癌は本邦22例目と極めてまれである. 本症は予後不良であるが, 自験例は1年6か月経過した現在も再発の兆候を認めず, CA19-9の上昇も認めていない.

Leiomyosarcoma of the inferior vena cava (IVC) is a rare neoplasm of venous smooth muscle. A case of leiomyosarcoma of the IVC which was successfully resected by early detection is described. A 63-year-old woman was seen at the hospital because of right abdominal pain, and abdominal ultrasonography revealed a 26×21mm sized tumor in S1 portion of the fiver. The correct diagnosis was not made even by computed tomographic scan, magnetic resonance imaging and transhepatic biopsy of the tumor. An exploratory laparotomy was carried out and the tumor was resected together with the hepatic IVC by using the femoro-axillary V-V bypass. We reconstructed the IVC by a EPTFE graft with rings. Pathologically this tumor was leiomyosarcoma of the IVC. The tumor was 4.0×2.5×2.0cm in size, protruded to the surface of the lumen of inferior vena cava, and its outside was adjacent of the liver through capsula fibrosa. The postoperative course was uneventful. There has been no sign of recurrence as of 6 months after the operation.

Our recent results on the synthesis and properties of a novel family of polysilsesquioxanes having oligothiophenes were reviewed. The polymers anchored on SiO2or ITO substrates showed excellent mechanical hardness due to the formation of a three-dimensional siloxane network structure and chemical linkage between polymer and the surface of metal-oxide substrates. Optical, electrochemical, and electrical properties of polymers were also investigated.

Recipients of spousal donor transplantation (SDT) have poorer histocompatibility and higher human leukocyte antigen (HLA) sensitization due to pregnancy than those receiving related donor transplantation (RDT). Thus, SDT carries a higher risk of acute rejection (AR). In our department, patients at a high immunological risk, such as those with ABO incompatibility and HLA sensitization, were considered for desensitization by double filtration plasmapheresis and preoperative administration of rituximab. In this study we compared the AR incidence rates between SDT and RDT according to their immunological risk. We performed RDT in 279 and SDT in 100 patients, a total of 379 cases, between 2000 and 2008; 48.7% of RDT and 67.0% of SDT cases were considered to be at a high immunological risk and underwent preoperative desensitization (P=0.002). Even though the AR incident rate of SDT was higher than RDT in the low immunological risk group, in which the patients had undergone transplantation without desensitization (RDT 24.4%, SDT 37.0%, P=0.012), there was no significant difference between the two donor type groups in the high immunological risk group, in which transplantation with desensitization occurred (RDT 21.3%, SDT 31.3%, P0.05). Preoperative administration of rituximab significantly reduced AR from 37.4% to 10.6% (P0.001), especially T-cell mediated rejection (36.4% to 20.2%, P=0.01). SDT no longer carries a high risk when appropriate desensitization, including the use of rituximab, is performed. Overall, the five-year graft survival rates were similar between RDT and SDT.

Recipients of spousal donor transplantation (SDT) have poorer histocompatibility and higher human leukocyte antigen (HLA) sensitization due to pregnancy than those receiving related donor transplantation (RDT). Thus, SDT carries a higher risk of acute rejection (AR). In our department, patients at a high immunological risk, such as those with ABO incompatibility and HLA sensitization, were considered for desensitization by double filtration plasmapheresis and preoperative administration of rituximab. In this study we compared the AR incidence rates between SDT and RDT according to their immunological risk. We performed RDT in 279 and SDT in 100 patients, a total of 379 cases, between 2000 and 2008; 48.7% of RDT and 67.0% of SDT cases were considered to be at a high immunological risk and underwent preoperative desensitization (P=0.002). Even though the AR incident rate of SDT was higher than RDT in the low immunological risk group, in which the patients had undergone transplantation without desensitization (RDT 24.4%, SDT 37.0%, P=0.012), there was no significant difference between the two donor type groups in the high immunological risk group, in which transplantation with desensitization occurred (RDT 21.3%, SDT 31.3%, P>0.05). Preoperative administration of rituximab significantly reduced AR from 37.4% to 10.6% (P

The unique immunologic environment of the liver, together with its anatomic location downstream of the gut, influences the maturation and function of its interstitial dendritic cell (DC) populations. These well-equipped, antigen-presenting cells play critical roles in regulation of innate and adaptive immunity. New information is emerging about the molecular regulation of liver DC maturation and function, and their tolerogenic potential, while new insight is being gained regarding interactions between liver DC and other immune effector cell populations (NK, NKT cells) in addition to T cells. During transplantation, factors that affect liver DC biology include ischemia-reperfusion injury, liver regeneration, viral infection and the actions of anti-inflammatory and immunosuppressive drugs. Herein, we review the molecular and cell biology of hepatic DC populations in relation to the regulation of alloimmune responses and liver transplant outcome.

An understanding of the mechanisms that suppress the human anti-pig cellular response is key for xenotransplantation. We have compared the ability of human regulatory T cells (Tregs) to suppress xenogeneic and allogeneic responses in vitro.Human peripheral blood mononuclear cells (PBMC), CD4+ T cells, or CD4+ CD25- T cells were stimulated with irradiated human or wild type (WT) or alpha1,3-galactosyltransferase gene-knockout (GT-KO) pig PBMC in the presence or absence of human CD4+ CD25 high Tregs. In separate experiments, 5- (and 6)-carboxyfluorescein diacetate succinimidyl ester-labeled human CD4+ T cells were stimulated with human or pig PBMC. The expansion and precursor frequencies of allo- and xenoreactive Tregs were assessed by labeling with FoxP3 mAb and flow cytometric analysis.The responses of human PBMC, CD4+ T cells, and CD4+ CD25- T cells to pig PBMC were stronger than to human PBMC (P0.05). Human anti-GT-KO responses were weaker than anti-WT responses (P0.05). Human CD4+ CD25 high Tregs suppressed proliferation of CD4+ CD25- T cells to both human and pig PBMC stimulator cells with the same efficiency. Alloreactive CD4+ CD25+ FoxP3 high responder T cells proliferated more than their xenoreactive counterparts (P0.05), although xenoreactive CD4+ CD25+ T cells proliferated more than alloreactive cells (P0.05). There was no difference in precursor frequency between allo- and xeno-reactive CD4+ CD25+ FoxP3 high cells.Human T-cell responses to pig cells are stronger than to allogeneic cells. The human response to GT-KO PBMC is weaker than to WT PBMC. Although human Tregs can suppress both responses, expansion of CD4+ CD25+ FoxP3 high cells against pig PBMC is weaker than against human PBMC. More human Tregs may be required to suppress the stronger xenogeneic response.

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-α), 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-α 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-α 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. (HEPATOLOGY 2008;48:1608–1620.)

Background/Aims The liver is comparatively rich in plasmacytoid (p) dendritic cells (DC), – innate immune effector cells that are also thought to play key roles in the induction and regulation of adaptive immunity. Methods Liver and spleen pDC were purified from fms-like tyrosine kinase ligand-treated control or lipopolysaccharide-injected C57BL/10 mice. Flow cytometric and molecular biologic assays were used to characterize their function and interaction with naturally occurring regulatory T cells (Treg). Results While IL-10 production was greater for freshly isolated liver compared with splenic pDC, the former produced less bioactive IL-12p70. Moreover, liver pDC expressed a low Delta4/Jagged1 Notch ligand ratio, skewed towards T helper 2 cell differentiation/cytokine production, and promoted allogeneic CD4 + T cell apoptosis. T cell proliferation in response to liver pDC was, however, enhanced by blocking IL-10 function at the initiation of cultures. In the absence of naturally occurring CD4 + CD25 + regulatory T cells, similar levels of T cell proliferation were induced by liver and spleen pDC and the pro-apoptotic activity of liver pDC was reversed. Conclusions The inferior T cell allostimulatory activity of in vivo -stimulated liver pDC may depend on the presence and function of Treg, a property that may contribute to inherent liver tolerogenicity.

It was recently reported that the induction of endotoxin tolerance (ET), which is defined as a reduced response to a lipopolysaccharide (LPS) challenge following the first LPS encounter, inhibits major histocompatibility complex (MHC)-restricted antigen presentation. This raises the question whether alloimmune responses can be inhibited by inducing ET in transplant donors. C57BL/6 mice were treated with a low dose of LPS prior to a challenge with a high dose of LPS to induce ET. Hearts from endotoxin-tolerized C57BL/6 mice were transplanted to BALB/c mice. The survival of the endotoxin-tolerized heart allografts was significantly prolonged. By using irradiated splenocytes from C57BL/6 mice and allogeneic splenocytes from BALB/c mice, a mixed lymphocyte reaction (MLR) assay was performed. The MLR assay used CFSE, and revealed that the splenocytes from the endotoxin-tolerized mice failed to induce the proliferation of allogeneic CD4(+) and CD8(+) T cells. Cytokine analyses of the supernatant of the MLR culture using endotoxin-tolerized stimulators revealed a distinct shift in the Th 1/Th 2 balance toward the Th 2-type response. The induction of ET increased the proportion of myeloid-related dendritic cells (DCs) expressing molecules necessary for antigen presentation, which favor the development of a Th 2 response; however, it reduced the proportion of lymphoid-related DCs expressing those molecules, which favor the development of the Th 1 response. Although the relevance of these findings with regard to the prolonged survival of the endotoxin-tolerized heart allografts remains to be elucidated, this is the first study to demonstrate that the induction of ET in donor animals inhibits alloimmune responses.