Clostridium butyricum-induced ω-3 fatty acid 18-HEPE elicits anti-influenza virus pneumonia effects through interferon-λ upregulation.

The precise mechanism by which butyrate-producing bacteria in the gut contribute to resistance to respiratory viral infections remains to be elucidated. Here, we describe a gut-lung axis mechanism and report that orally administered Clostridium butyricum (CB) enhances influenza virus infection resistance through upregulation of interferon (IFN)-λ in lung epithelial cells. Gut microbiome-induced ω-3 fatty acid 18-hydroxy eicosapentaenoic acid (18-HEPE) promotes IFN-λ production through the G protein-coupled receptor (GPR)120 and IFN regulatory factor (IRF)-1/-7 activations. CB promotes 18-HEPE production in the gut and enhances ω-3 fatty acid sensitivity in the lungs by promoting GPR120 expression. This study finds a gut-lung axis mechanism and provides insights into the treatments and prophylaxis for viral respiratory infections.

Clostridium butyricum enhances colonization resistance against Clostridioides difficile by metabolic and immune modulation.

Clostridioides difficile infection (CDI) represents the leading cause of nosocomial diarrhea worldwide and is associated with gut dysbiosis and intestinal damage. Clostridium butyricum MIYAIRI 588 (CBM 588) contributes significantly to reduce epithelial damage. However, the impacts of CBM 588 on antibacterial therapy for CDI are not clear. Here we show that CBM 588 enhanced the antibacterial activity of fidaxomicin against C. difficile and negatively modulated gut succinate levels to prevent C. difficile proliferation and downregulate tumor necrosis factor-α (TNF-α) producing macrophages in the colon lumina propria (cLP), resulting in a significant decrease in colon epithelial damage. Additionally, CBM 588 upregulated T cell-dependent pathogen specific immunoglobulin A (IgA) via interleukin (IL)-17A producing CD4+ cells and plasma B cells in the cLP, and Th17 cells in the cLP enhanced the gut epithelial barrier function. IL-17A and succinic acid modulations with CBM 588 enhance gut colonization resistance to C. difficile and protect the colon tissue from CDI.

Improved detection of donor-specific HLA-class II antibody in kidney transplant recipients by modified immunocomplex capture fluorescence analysis.

Immunocomplex capture fluorescence analysis (ICFA) which basic principle is same as Luminex crossmatch (LXM), could detect donor-specific HLA antibody (DSA). The advantages of ICFA are (i) detection of DSA and (ii) no requirement of viable cells over the flow cytometry crossmatch (FCXM). However, FCXM has been widely used because of its higher sensitivity than ICFA, in particular HLA-class II antibody detection. In this study the accuracy of DSA detection against HLA-class II was investigated by modifying the original method of ICFA. Increment of the sensitivity was found when purified peripheral blood mononuclear cells (PBMCs) were used instead of whole blood. An ICFA-PBMC in addition to FCXM-T/B was conducted for 118 patients before kidney transplantation and 13 patients with de novo DSA against HLA-class II after transplantation. Significantly positive correlation was observed between the values of ICFA-PBMC and DSA mean fluorescence intensity (MFI) targeting class II (p < 0.0001). When the cutoff level of 1.4 was determined by receiver operating characteristic curve analysis, the average DSA MFI was found to be significantly higher in the ICFA-PBMC (class II) positive group comparing to that in the negative group (12,217 vs 3885, p = 0.0027). ICFA-PBMC and optimized cutoff level could provide valid information in cases of suspected DSA.

Clinical Significance of Shared T Cell Epitope Analysis in Early De Novo Donor-Specific Anti-HLA Antibody Production After Kidney Transplantation and Comparison With Shared B cell Epitope Analysis.

In pre-sensitizing events, immunological memory is mainly created via indirect allorecognition where CD4+ T cells recognize foreign peptides in the context of self-HLA class II (pHLA) presented on antigen-presenting cells. This recognition makes it possible for naive CD4+ T-helper cells to differentiate into memory cells, resulting in the creation of further antibody memory. These responses contribute to effective secretion of donor-specific anti-HLA antibodies (DSA) after second encounters with the same peptide. Preformed donor-reactive CD4+ memory T cells may induce early immune responses after transplantation; however, the tools to evaluate them are limited. This study evaluated shared T cell epitopes (TEs) between the pre-sensitizing and donor HLA using an in silico assay, an alternative to estimate donor-reactive CD4+ memory T cells before transplantation. In 578 living donor kidney transplants without preformed DSA, 69 patients had anti-HLA antibodies before transplantation. Of them, 40 had shared TEs and were estimated to have donor-reactive CD4+ memory T cells. De novo DSA formation in the early phase was significantly higher in the shared TE-positive group than in the anti-HLA antibody- and shared TE-negative groups (p=0.001 and p=0.02, respectively). In conclusion, evaluation of shared TEs for estimating preformed donor-reactive CD4+ memory T cells may help predict the risk of early de novo DSA formation after kidney transplantation.

Assessment of the effect of thinning on the resistance of Pinus thunbergii Parlat. trees in mature coastal forests to tsunami fluid forces.

The Great East Japan Tsunami, triggered by the earthquake that occurred on March 11, 2011 in the Pacific Ocean, caused significant fatalities and socioeconomic damage. As recovery of a disaster area requires significant time, all possible mitigation measures must be prepared in advance for future events. As a tsunami countermeasure, coastal forests have been acknowledged to considerably reduce tsunami energy and decrease tsunami-related damage. In the Great East Japan tsunami, many trees of coastal forests were damaged by trunk breakage and overturning. This led to further infrastructural damage as the debris were transported landward and seaward by floodwaters. To better protect coastal areas from the secondary effects of tsunamis and reduce tsunami energy, coastal forests must exhibit higher resistance. This research investigated the effect of forestry management by applying different levels of thinning of trees as a means of resistance to tree damage under tsunami events. In October of 1999, study plots were established with different thinning intensities in a mature coastal forest of Pinus thunbergii trees. As a useful indicator of the resistance of coastal forests to tsunamis, the threshold tsunami velocities at which trees in these study plots begin to be destroyed were calculated using a mechanistic model. The results revealed that trunk diameter is the most important parameter for increasing resistance to tsunamis. An analysis of the generalized linear model for diameter growth showed that heavy thinning best enhanced the diameter growth. Therefore, heavy thinning is the most effective approach to increasing the resistance of trees to tsunamis. Considering the relationship between resistance to tsunami and inundation depth, the resistance to tsunami decreased rapidly with increasing inundation depth in all plots. Differences in the resistance to the tsunami were not observed across all plots when the inundation depth exceeded the mean tree height.

Clostridium butyricum MIYAIRI 588-Induced Protectin D1 Has an Anti-inflammatory Effect on Antibiotic-Induced Intestinal Disorder.

Metabolites are thought as the end products in cellular regulatory processes and their levels show the strongest relationships with the phenotype. Previously, we showed that the administration of Clostridium butyricum MIYAIRI 588 (CBM 588) upregulated protectin D1, an anti-inflammatory lipid metabolite, in colon tissue under antibiotic therapy. However, how CBM 588 induces protectin D1 expression and whether the metabolite has anti-inflammatory effects on antibiotic-induced inflammation are unclear. Therefore, here, we evaluated the effect of CBM 588 on lipid metabolism and protectin D1 in gut protection from antibiotic-induced intestinal disorders. In the CBM 588 treatment group, expression levels of genes encoding lipid receptors related to the conversion of DHA to protectin D1, such as polyunsaturated fatty acid (PUFA) receptors, G-protein coupled receptor 120 (GPR120), and 15-lipoxygenase (LOX), were increased in colon tissue. CD4+ cells producing interleukin (IL)-4, the main component of T helper type 2 (Th2) cells that can activate 15-LOX, also increased in CBM 588-treated groups even after clindamycin co-administration. In addition, similar to CBM 588, exogenously administered protectin D1 reduced inflammatory cytokines, while IL-10 and TGF-ß1, works as anti-inflammatory cytokines, were increased. Our data revealed that CBM 588 activated 15-LOX to enhance protectin D1 production by increasing IL-4-producing CD4+ cell population in the intestinal tract. Additionally, CBM 588-induced protectin D1 clearly upregulated IL-10-producing CD4+ cells to control antibiotic-induced gut inflammation. We provide new insights into CBM 588-mediated lipid metabolism induction for the treatment of gut inflammatory diseases.

Analysis of T and B Cell Epitopes to Predict the Risk of de novo Donor-Specific Antibody (DSA) Production After Kidney Transplantation: A Two-Center Retrospective Cohort Study.

Risk prediction of de novo donor specific antibody (DSA) would be very important for long term graft outcome after organ transplantation. The purpose of this study was to elucidate the association of eplet mismatches and predicted indirectly recognizable HLA epitopes (PIRCHE) scores with de novo DSA production. Our retrospective cohort study enrolled 691 living donor kidney transplantations. HLA-A, B, DRB and DQB eplet mismatches and PIRCHE scores (4 digit of HLA-A, B, DR, and DQ) were determined by HLA matchmaker (ver 2.1) and PIRCHE-II Matching Service, respectively. Weak correlation between eplet mismatches and PIRCHE scores was identified, although both measurements were associated with classical HLA mismatches. Class II (DRB+DQB) eplet mismatches were significantly correlated with the incidence of de novo class II (DR/DQ) DSA production [8/235 (3.4%) in eplet mismatch ≤ 13 vs. 92/456 (20.2%) in eplet mismatch ≥ 14, p < 0.001]. PIRCHE scores were also significantly correlated with de novo class II DSA production [26/318 (8.2%) in PIRCHE ≤ 175 vs. 74/373 (19.8%) in PIRCHE ≥ 176, p < 0.001]. Patients with low levels of both class II eplet mismatches and PIRCHE scores developed de novo class II DSA only in 4/179 (2.2%). Analysis of T cell and B cell epitopes can provide a beneficial information on the design of individualized immunosuppression regimens for prevention of de novo DSA production after kidney transplantation.

Clostridium butyricum Modulates the Microbiome to Protect Intestinal Barrier Function in Mice with Antibiotic-Induced Dysbiosis.

Clostridium butyricum MIYAIRI 588 (CBM 588) is a probiotic bacterium that has previously been used to prevent antibiotic-associated diarrhea. However, the underlying mechanism by which CBM 588 protects the gut epithelial barrier remains unclear. Here, we show that CBM 588 increased the abundance of Bifidobacterium, Lactobacillus, and Lactococcus species in the gut microbiome and also enhanced the intestinal barrier function of mice with antibiotic-induced dysbiosis. Additionally, CBM 588 significantly promoted the expansion of IL-17A-producing γδT cells and IL-17A-producing CD4 cells in the colonic lamina propria (cLP), which was closely associated with changes in the intestinal microbial composition. Additionally, CBM 588 plays an important role in controlling antibiotic-induced gut inflammation through upregulation of anti-inflammatory lipid metabolites such as palmitoleic acid, 15d-prostaglandin J2, and protectin D1. This study reveals a previously unrecognized mechanism of CBM 588 and provides new insights into gut epithelial barrier protection with probiotics under conditions of antibiotic-induced dysbiosis.

Suppressive Effect of Everolimus on IL-2, IL-10, IL-21, and IFNγ Levels: Implications for the Successful Minimization of Calcineurin Inhibitor Use in Transplantation.

BACKGROUND: Success with calcineurin inhibitors (CNIs) such as cyclosporine A (CSA) and tacrolimus (TAC) in organ transplantation has demonstrated that cytokine suppression is a key factor in patient management. However, the exact effects of recently introduced immunosuppressive agents other than CNI on cytokine expression remain unknown. In this study, the action of the mTOR-inhibitor everolimus (EVR) and that of the antimetabolite mycophenolic acid (MPA) on the transcription of several cytokines was investigated. METHODS: Peripheral blood mononuclear cells obtained from healthy volunteers were stimulated with anti-CD3/28 microbeads in the presence of CSA, TAC, EVR, and/or MPA for 8 hours. The mRNA levels of each cytokine were measured using quantitative real-time polymerase chain reaction. RESULTS: MPA had no inhibitory effect on any of the cytokines tested. EVR showed moderate inhibition of IL-2, IL-10, IL-21, and IFNγ levels. These cytokines were further analyzed to investigate the additive effect of EVR in combination with CNI. The beneficial effect of EVR addition was seen at low concentrations of CSA or TAC, while no additive effect was observed at high concentrations. CONCLUSIONS: EVR might effectively inhibit the activation of recipient immune cells in combination with a low dose of CNI, maximizing clinical benefit by preventing graft rejection and alleviating CNI-induced adverse effects.

Lower incidence of de novo donor-specific antibodies against HLA-DR in ABO-incompatible renal transplantation.

Recently, in vitro experiments have demonstrated that anti-blood group A/B antibody binding to endothelial cells induce a protective effect against antibody-mediated injury. This study aimed to clarify the potential clinical benefit of ABO incompatibility in donor-specific HLA antibody (DSA)-induced chronic antibody-mediated rejection (ABMR). We enrolled 215 ABO-incompatible renal transplant (ABO-I) and 467 ABO-identical/compatible renal transplant recipients (ABO-Id/C). The prevalence of de novo DSA production and incidence of biopsy-proven chronic ABMR were compared between the two groups. The incidence of DR-associated de novo DSA was significantly lower in ABO-I than in ABO-Id/C (P = 0.028). Diagnostic biopsy for ABMR was conducted in 54 patients (11 ABO-I and 43 ABO-Id/C). Biopsy-proven chronic ABMR was lower in ABO-I than in ABO-Id/C (27.3% [3/11] vs. 44.2% [19/43]) patients. Our findings suggest that ABO incompatibility may cause low production of DR-associated de novo DSA, possibly resulting in a reduced incidence of chronic ABMR.

Increased CD40L+PD-1+ follicular helper T cells (Tfh) as a biomarker for predicting calcineurin inhibitor sensitivity against Tfh-mediated B-cell activation/antibody production after kidney transplantation.

It is unclear to what extent the development of follicular helper T cells (Tfh) and de novo donor-specific human leukocyte antigen antibody (DSA) production could be influenced by immunosuppressive agents, particularly calcineurin inhibitor (CNI; cyclosporine or tacrolimus), after kidney transplantation. Here, the effects of immunosuppressive agents on Tfh-mediated B-cell activation and antibody production were investigated. In vitro circulating Tfh (cTfh; memory CD4+CXCR5+)/B-cell (CD19+) co-culture assays revealed that CNI considerably inhibited cTfh-mediated B-cell activation and IgG antibody secretion through the suppression of IL-21 and IL-2. Both IL-21 and CD40L up-regulated IL-2 receptors (CD25) on B cells, and anti-CD25 antibody induced apoptosis of activated B cells, resulting in the inhibition of IgG production. The frequency of cTfh-expressed CD40L and PD-1 was elevated in patients with de novo DSA 1 year after transplantation. The degree of inhibition by CNI was dependent on Staphylococcal enterotoxin B-induced CD40L+PD-1+ cTfh up-regulation level. Our data demonstrate that CD40L+PD-1+cTfh could be a marker to implicate individual difference in CNI sensitivity for Tfh-mediated B-cell activation in kidney transplantation.

Peripheral blood immune response-related gene analysis for evaluating the potential risk of chronic antibody-mediated rejection.

Noninvasive methods for the early diagnosis of chronic antibody-mediated rejection (cAMR) are desired for patients with de novo (dn) donor-specific HLA antibody (DSA). This study aimed to elucidate the clinical relevance of immune-related gene expression in peripheral blood of kidney transplant recipients. The expression levels of fourteen key molecules (Foxp3, CTLA-4, CCR7, TGF-ß, IGLL-1, IL-10, ITCH, CBLB, Bcl-6, CXCR5, granzyme B, CIITA, Baff, TOAG-1/TCAIM) related to regulatory/cytotoxic function of immune cells were compared in 93 patients, which were divided into Groups A (clinical cAMR with dn DSA, n = 16), B (subclinical cAMR with dn DSA, n = 17), C (negative cAMR with dn DSA, n = 21) and D (stable function without dn DSA, n = 39). CIITA mRNA expression levels in groups B and C were significantly lower than those in group D (p < 0.01). Moreover, the CTLA-4 mRNA expression in group A was significantly higher than that in groups B and C (p < 0.01). ROC curve analysis suggested that CIITA (AUC = 0.902) and CTLA-4 (AUC = 0.785) may serve as valuable biomarkers of the stage of dn DSA production and clinical cAMR, respectively. In addition to dn DSA screening, monitoring of CIITA and CTLA-4 in peripheral blood could offer useful information on the time course of the development of cAMR.

Favorable results in ABO-incompatible renal transplantation without B cell-targeted therapy: Advantages and disadvantages of rituximab pretreatment.

The effectiveness of desensitization with rituximab in ABO-incompatible renal transplantation (ABO-I) has been widely reported. However, ABO-I outcomes are still worse than those of ABO-identical or ABO-compatible renal transplantation (ABO-Id/C). We retrospectively examined the outcomes in consecutive living donor ABO-Id/C (n = 412) and ABO-I (n = 205) cases to elucidate the causes of inferiority in ABO-I. ABO-I cases included recipients treated with rituximab (RIT, n = 131), splenectomy (SPX, n = 21), or neither because of low anti-A/B antibody titers (NoR/S, n = 53). Graft survival, infection, and de novo HLA antibody production were compared for ABO-I and ABO-Id/C, followed by stratification into RIT and NoR/S groups. Propensity score-based methods were employed to limit selection bias and potential confounders. Overall graft survival for ABO-I was significantly lower than that for ABO-Id/C (92.8% vs 97.2% after 5 years, P = .0037). Graft loss due to infection with ABO-I was significantly more frequent than that with ABO-Id/C, whereas acute antibody-mediated rejection (AMR) caused no graft failure in ABO-I recipients. Stratified analysis demonstrated significantly higher infection risk with RIT than with NoR/S. Safe reduction or avoidance of rituximab in desensitization protocols might contribute to further improvement of ABO-I outcome.

MiR-142-5p and miR-486-5p as biomarkers for early detection of chronic antibody-mediated rejection in kidney transplantation.

De novo donor-specific HLA antibody (DSA) would not necessarily contribute to chronic antibody-mediated rejection (CAMR) in kidney transplantation. Here, we investigated whether PBMC miRNAs could be predictable biomarkers for CAMR. Microarray profiling of 435 mature miRNAs in pooled samples was conducted. Individual analysis revealed that miR-142-5p was significantly (p < 0.01) underexpressed in patients with DSA. After DSA production, miR-486-5p and its target PTEN/foxO3 mRNA were significantly overexpressed (p < 0.01) and underexpressed (p < 0.01), respectively, in patients with biopsy-proven CAMR, compared with non-CAMR. Our studies suggest that miRNA expression patterns may serve as noninvasive diagnostic biomarkers to evaluate immune response and kidney allograft status.

Negative regulation of HLA-DR expression on endothelial cells by anti-blood group A/B antibody ligation and mTOR inhibition.

Donor-specific antibody (DSA), particularly against HLA class II, is a major cause of chronic antibody-mediated rejection (CAMR) after transplantation, although ABO-incompatible kidney transplantation has recently demonstrated favorable graft outcomes. The condition of no injury even in the presence of anti-donor antibody has been referred to as "accommodation", which would be one of the key factors for successful long-term graft survival. The purpose of this study was to analyze the beneficial effect of anti-blood group A/B antibody ligation on endothelial cells against HLA-DR antibody-mediated, complement-dependent cytotoxicity (CDC). Blood group A/B-expressing endothelial cells EA.hy926 or Human Umbilical Vein Endothelia Cells (HUVEC) were incubated with IFNγ in the presence or absence of anti-blood group A/B antibody or mTOR inhibitor (mTOR-i) for 48h. The effects on signaling pathway, HLA expression, complement regulatory factors, and CDC were investigated. Expression of HLA-DR on EA.hy926 or HUVEC were successfully elicited by IFNγ treatment, although little or no expression was observed in quiescent cells. Pre-incubation with anti-blood group A/B antibody had resistance to HLA-DR antibody-mediated CDC against IFNγ-treated cells in a concentration-dependent manner. This finding was ascribed to decreased expression of HLA-DR by post-translational regulation and increased expression of CD55/59, which was related to ERK and mTOR pathway inhibition. mTOR-i also inhibited HLA-DR expression by itself. Furthermore, the combination of mTOR-I and anti-blood group A/B ligation had an additive effect in preventing HLA-DR antibody-mediated CDC. Anti-blood group A/B antibody might play a preventive role in CAMR. Inhibition of the ERK and mTOR pathways may contribute to the development of a novel treatment in the maintenance period after transplantation.

5-year follow-up of a randomized clinical study comparing everolimus plus reduced-dose cyclosporine with mycophenolate mofetil plus standard-dose cyclosporine in de novo kidney transplantation: Retrospective single center assessment.

The aim of this study is to evaluate the efficacy and safety of everolimus plus reduced-dose cyclosporine compared with mycophenolate mofetil plus standard-dose cyclosporine 5years after living donor kidney transplantation. Between March 2008 and August 2009, 24 living donor kidney transplantations were enrolled in a 2-year, multicenter, randomized phase 3 study (RAD001A1202 study). 24 recipients were randomly classified into two groups and closely observed for 5years. 13 recipients were administered steroid, reduced-dose cyclosporine, everolimus and basiliximab (EVR group). 11 recipients were administered steroid, standard-dose cyclosporine, mycophenolate mofetil and basiliximab (STD group). Two groups were compared not only in graft function including estimated glomerular filtration rate (eGFR), and proteinuria, but also in adverse events such as de novo donor-specific antibody (DSA) production, rejection, new-onset diabetes, hyperlipidemia, and cytomegalovirus (CMV) infection. No graft loss was identified in 5years. The incidences of acute T cell rejection, de novo DSA production, hyperlipidemia, and new-onset diabetes were similar. eGFR levels throughout the observation periods were similar. Three cases of proteinuria were identified in STD group. One case of proteinuria observed in EVR group was well controlled with angiotensin receptor blocker. Incidence of CMV infection in CMV antibody-positive recipients was significantly lower in EVR group. The safety and efficacy of reduced-dose cyclosporine and everolimus protocol were similar to those of standard-dose cyclosporine and mycophenolate mofetil other than for superior prevention of CMV infection.

Changes in ABCB1 mRNA Expression in Peripheral Blood Cells before and after Renal Transplantation.

Cyclosporine (CSA), which is one of the substrates of ATP binding cassette subfamily B member 1 (ABCB1), is widely used as an immunosuppressant in patients undergoing transplantation. The expression level of P-glycoprotein on lymphocytes that is encoded by ABCB1 gene is considered to be one of the major causes of differences in intracellular CSA concentration. The clinical relevance of ABCB1 mRNA expression in peripheral blood was analyzed. We examined (i) the relationship between ABCB1 mRNA and the intracellular concentration of CSA in vitro, (ii) the change in long-term ABCB1 mRNA expression levels, and (iii) its association with acute rejection (AR) or cytomegalovirus (CMV) reactivation in living-donor renal transplantation. A significantly negative correlation between ABCB1 mRNA expression and intracellular CSA concentration in vitro was obtained (p<0.05). ABCB1 mRNA expression was significantly reduced (55%) 1 week after transplantation (p<0.001) and returned to the pre-transplantation level after 1 year. Although the sample size may be too small to obtain a definitive conclusion, no association was observed between ABCB1 mRNA expression levels and AR or CMV reactivation.

Functional difference between membrane-bound and soluble human thrombomodulin.

BACKGROUND: For successful xenotransplantation, in addition to α1,3-galactosyltransferase gene-knockout and human complement regulatory protein (CD46, CD55, CD59) gene insertion, cloned pigs expressing human thrombomodulin (hTM) have been produced to solve the problem of molecular incompatibility in their coagulation system. Recombinant soluble hTM (S-hTM) which has been recently approved for treatment of disseminated intravascular coagulation might be potentially available. The purpose of this study is to examine the functional difference in endothelial cells between membrane-bound hTM (MB-hTM) and S-hTM and to elucidate effective strategy using both types of hTM. METHODS: The following factors regarding coagulation and inflammation were compared between hTM-expressing pig aortic endothelial cells (PAEC) derived from cloned pig and nontransgenic PAEC in the presence of S-hTM under tumor necrosis factor-α-activated conditions; (i) clotting time (ii) pig tissue factor (TF), (iii) pig E-selectin, (iv) direct prothrombinase activity, (v) activated protein C (APC), and (vi) prothrombinase activity. RESULTS: The MB-hTM significantly suppressed the expression of pig TF and E-selectin and direct prothrombinase activity in tumor necrosis factor-α-activated PAEC, suggesting strong anti-inflammatory effect, compared to S-hTM. In contrast, S-hTM had more potent capacity to inhibit thrombin generation and to produce APC than MB-hTM, although MB-hTM had the same level of capacity as human endothelial cells. CONCLUSIONS: It was speculated that S-hTM treatment would be of assistance during high-risk periods for excessive thrombin formation (e.g., ischemia reperfusion injury or severe infection/rejection). Considering the properties of MB-hTM exhibiting anti-inflammatory function as well as APC production, hTM-expressing cloned pigs might be indispensible to long-term stabilization of graft endothelial cells.

Clinical relevance of post-transplant pharmacodynamic analysis of cyclosporine in renal transplantation.

Although therapeutic drug monitoring based on blood concentration has been widely implemented in transplant recipients treated with immunosuppressive agents, clinical adverse events such as rejection, infection or drug-induced toxicity caused by inappropriate dosage cannot be completely controlled. Development of an effective assay for optimized immunosuppression would be desirable, which can potentially lead to personalized medicine in renal transplantation. Cyclosporine (CSA) pharmacodynamic analysis using carboxyfluorescein diacetate succinimidyl ester (CFSE)-based T cell proliferation assay was examined in 66 kidney transplant recipients before and after transplantation. Two parameters, the 50% inhibitory concentration (IC50) and the percentage of T-cell proliferation values at the lower plateau (bottom), were compared with clinical events. A significant relation in CSA pharmacodynamic parameters was observed between pre- and post-transplantation. Analysis of the association between clinical outcomes and pharmacodynamic parameters in post-transplant samples demonstrated the following findings: (i) cytomegalovirus (CMV)/varicella zoster virus (VZV) reactivation and CSA-induced nephrotoxicity were significantly associated with high sensitivity to CSA (low bottom or low IC50), (ii) acute T cell-mediated rejection (ATMR) was significantly related to low sensitivity to CSA (high bottom), and (iii) de novo human leukocyte antigen (HLA) antibody production was associated with lower bottom and IC50 values, although the elucidation of those mechanisms is still in progress. It was suggested that CSA pharmacodynamics applied at post-transplantation would be useful for optimizing immunosuppressive therapy.

Influence of olive-derived hydroxytyrosol on the toll-like receptor 4-dependent inflammatory response of mouse peritoneal macrophages.

Macrophages play important roles in the host innate immune response and are involved in the onset of diseases caused by inflammation. Toll-like receptor 4 (TLR4)-mediated inflammatory responses of macrophages may be associated with diseases such as diabetes and diseases of the cardiovascular system. Hydroxytyrosol (HT) exerts strong antioxidant and anti-inflammatory effects and may be applied in the treatment of inflammatory diseases. In the present study conducted in vitro, we investigated the effects of the TLR4-dependent anti-inflammatory effect of HT on peritoneal macrophage of BALB/c mice. We show here that the elevated levels of iNOS gene expression and nitric oxide production induced by lipopolysaccharide (LPS) (0.25 µg/ml) were suppressed by HT (12.5 µg/ml). LPS-dependent NF-κB gene expression and phosphorylation of NF-κB were not affected by HT under these conditions. In contrast, the expression of TNF-α was significantly increased in the presence of LPS and HT. These results suggest that HT suppressed nitric oxide production by decreasing iNOS gene expression through a mechanism independent of the NF-κB signaling pathway. These novel findings suggest that the modulation by HT of the expression of genes involved in inflammation may involve multiple mechanisms.