A Novel Method of CD31-Combined ABO Carbohydrate Antigen Microarray Predicts Acute Antibody-Mediated Rejection in ABO-Incompatible Kidney Transplantation.

Isohemagglutinin assays employing red blood cells (RBCs) are the most common assays used to measure antibody titer in ABO-incompatible kidney transplantation (ABOi KTx). However, ABO antigens expressed on RBCs are not identical to those of kidney and antibody titers do not always correlate with clinical outcome. We previously reported that CD31 was the main protein linked to ABO antigens on kidney endothelial cells (KECs), which was different from those on RBCs. We developed a new method to measure antibody titer using a microarray of recombinant CD31 (rCD31) linked to ABO antigens (CD31-ABO microarray). Mass spectrometry analysis suggested that rCD31 and native CD31 purified from human kidney had similar ABO glycan. To confirm clinical use of CD31-ABO microarray, a total of 252 plasma samples including volunteers, hemodialysis patients, and transplant recipients were examined. In transplant recipients, any initial IgG or IgM antibody intensity >30,000 against the donor blood type in the CD31-ABO microarray showed higher sensitivity, specificity, positive predictive value, and negative predictive value of AABMR, compared to isohemagglutinin assays. Use of a CD31-ABO microarray to determine antibody titer specifically against ABO antigens expressed on KECs will contribute to precisely predicting AABMR or preventing over immunosuppression following ABOi KTx.

ANGIOGRAPHIC RISK FACTORS FOR RECURRENCE OF MACULAR EDEMA ASSOCIATED WITH BRANCH RETINAL VEIN OCCLUSION.

PURPOSE: To examine angiographic risk factors for the recurrence of macular edema associated with branch retinal vein occlusion. METHODS: We consecutively included 51 patients with treatment-naive branch retinal vein occlusion involving the macular area. Each eye initially received 3 monthly ranibizumab injections, with additional injections as necessary. At Month 3, we examined parafoveal vessel diameter indexes (VDI) in all sectors using optical coherence tomography angiography and determined the association with retinal thickness changes (Month 3-Month 5) and the number of ranibizumab injections during 12 months. RESULTS: Parafoveal VDIs in the affected, nasal, and temporal sectors at Month 3 were significantly associated with corresponding parafoveal thickening (P = 0.020, 0.010, and <0.001, respectively), and the parafoveal VDIs in the affected and temporal sectors were significantly associated with future foveal thickening (P = 0.037, and 0.026, respectively). Moreover, the parafoveal VDI in the temporal sector showed a significant association with the total required number of ranibizumab injections (P = 0.040). CONCLUSION: The parafoveal VDI may adequately represent the degree of congestion associated with branch retinal vein occlusion. Particularly, the VDI in the temporal sector may be a good predictor of future retinal thickening in the corresponding parafovea and the fovea and the number of ranibizumab injections.

NONPERFUSION AREA QUANTIFICATION IN BRANCH RETINAL VEIN OCCLUSION: A Widefield Optical Coherence Tomography Angiography Study.

PURPOSE: To precisely quantify retinal nonperfusion areas (NPAs) in branch retinal vein occlusion using widefield optical coherence tomography angiography (OCTA) and examine their association with neovascular complications. METHODS: We enrolled 26 patients with treatment-naïve branch retinal vein occlusion and prospectively examined them for 12 months. After 3 monthly ranibizumab injections to treat macular edema, each patient underwent ultra-widefield (UWF) fluorescein angiography (FA) and OCTA. Ultra-widefield FA was additionally performed at Month 12. For UWF FA, the retinal NPA was measured using the equipment's built-in software. For OCTA, we used panoramic image montaged from 5 single 12 × 12 mm2 images and quantified the retinal NPA using a Gullstrand eye with a grid scale at each patient. Measurements were expressed in terms of actual values and disc area units. RESULTS: The retinal NPAs as measured using single OCTA and panoramic OCTA were significantly associated with that measured using UWF FA (P < 0.001 for both). Retinal neovascularization lesions were observed in 4 (15.4%) of 26 eyes. For patients with accompanying neovascularization, the retinal NPA measured using UWF FA, single OCTA, and panoramic OCTA were 187.9 ± 39.5 mm2 (109.9 ± 21.4 disc area), 34.3 ± 13.7 mm2 (19.9 ± 7.7 disc area), and 106.6 ± 24.5 mm2 (62.4 ± 13.6 disc area), respectively, which were larger than for those without neovascularization (P < 0.001, 0.014, and <0.001, respectively). CONCLUSION: Using widefield OCTA, we could quantify the retinal NPA of eyes with branch retinal vein occlusion. These could serve as valid references to assess the risk of neovascular complications.

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.

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.

FIXATION STATUS AFTER RESOLUTION OF MACULAR EDEMA ASSOCIATED WITH BRANCH RETINAL VEIN OCCLUSION.

PURPOSE: To evaluate fixation status of eyes with branch retinal vein occlusion (BRVO) and to investigate its association with other clinical parameters. METHODS: This study included 57 consecutive eyes with BRVO after resolution of macular edema. Fixation status was determined by microperimetry. Defect length of the foveal ellipsoid zone band was measured by optical coherence tomography, and retinal perfusion status was assessed by optical coherence tomography angiography. RESULTS: In microperimetry, the mean fixation rate around the gravitational center of all fixation points (defined as the fixation center) was found to be 79.8 ± 18.9%, which was significantly associated with defect length of the foveal ellipsoid zone band (P < 0.001) and distance between the foveal and fixation centers (P = 0.012). The integrity of the ellipsoid zone band at the fixation center was intact in 55 eyes (96.5%). Fixation centers were located within and outside the foveal avascular zone in 33 (57.9%) and 24 (42.1%) eyes, respectively; among the latter eyes, all fixation centers were perfused. Downward deviation of fixation points was rare, despite variations in the occluded area; there was a significant difference in distribution of deviation between eyes with superotemporal and inferotemporal BRVO (P < 0.001). CONCLUSION: In eyes with BRVO, fixation status was strongly associated with visual acuity, morphologic damage, and retinal perfusion status both in the foveal area and at the fixation center after resolution of macular edema. This information regarding fixation status could facilitate vision management in patients with BRVO.

EVALUATION OF MACULAR ISCHEMIA IN EYES WITH BRANCH RETINAL VEIN OCCLUSION: An Optical Coherence Tomography Angiography Study.

PURPOSE: To quantitatively assess macular morphology and perfusion status using optical coherence tomography, and optical coherence tomography angiography in eyes with branch retinal vein occlusion when macular edema has completely resolved, and to investigate the impact on visual function. METHODS: Thirty consecutive eyes with branch retinal vein occlusion-macular edema that resolved after treatment with intravitreal ranibizumab injections were included. Macular sensitivity was measured by microperimetry; defect length of foveal ellipsoid zone band was measured using optical coherence tomography; foveal avascular zone and parafoveal nonperfusion areas (NPA) were measured by optical coherence tomography angiography. RESULTS: The logarithm of minimum angle of resolution visual acuity was significantly associated with the defect length of the foveal ellipsoid zone band (P = 0.005), the parafoveal NPA in the superficial capillary plexus (P = 0.007), and the parafoveal NPA in the deep capillary plexus (P = 0.006). Macular sensitivity correlated with parafoveal thickness on the affected side (P = 0.034), the defect length of the foveal ellipsoid zone band (P = 0.048), parafoveal NPA in the superficial capillary plexus (P = 0.008), and parafoveal NPA in the deep capillary plexus (P = 0.012). Multivariate analysis where the only significant parameters in the univariate analyses were used as the independent variables showed that parafoveal NPA was most significantly associated with the logarithm of minimum angle of resolution visual acuity (ß = 0.500, P = 0.005) and macular sensitivity (ß = -0.480, P = 0.007). CONCLUSION: In eyes with branch retinal vein occlusion-macular edema resolved by intravitreal ranibizumab treatments, visual function was strongly associated with parafoveal NPA size.

EVALUATION OF MACULAR ISCHEMIA IN EYES WITH CENTRAL RETINAL VEIN OCCLUSION: An Optical Coherence Tomography Angiography Study.

PURPOSE: To quantitatively assess macular perfusion status using optical coherence tomography angiography in eyes with aflibercept-treated central retinal vein occlusion and resolved macular edema and to investigate the impact of macular morphology and perfusion status on visual function. METHODS: This prospective consecutive case series included 23 patients with central retinal vein occlusion. All patients received intravitreal aflibercept injections before analysis. Visual acuity, macular sensitivity, and the macular nonperfusion area (NPA) were evaluated in eyes without macular edema. The macular NPA was evaluated by optical coherence tomography angiography using 3 mm × 3 mm images of the macula. Foveal ellipsoid zone disruption was also analyzed. RESULTS: The superficial macular NPA measured 4.15 mm ± 0.71 mm (95% confidence interval 3.85-4.46), and the deep macular NPA measured 4.23 mm ± 0.97 mm (95% confidence interval 3.82-4.56). The logarithm of the minimum angle of resolution visual acuity was significantly associated with foveal ellipsoid zone disruption (P = 0.001), the superficial macular NPA (P = 0.015), and the deep macular NPA (P = 0.018). Macular sensitivity correlated negatively with logarithm of the minimum angle of resolution visual acuity (P = 0.007), the superficial macular NPA (P = 0.029), and the deep macular NPA (P = 0.040), but not with the foveal ellipsoid zone disruption (P = 0.435). CONCLUSION: Optical coherence tomography angiography is a novel technique that enables segmented evaluation of the macular perfusion status in eyes with central retinal vein occlusion and provides visual prognostic information. Enlargement of the macular NPA in the superficial and deep layers was significantly correlated with impaired visual acuity and with decreased macular sensitivity in patients with aflibercept-treated central retinal vein occlusion and resolved macular edema.

Anti-Vascular Endothelial Growth Factor Therapy for Macular Edema following Central Retinal Vein Occlusion: 1 Initial Injection versus 3 Monthly Injections.

PURPOSE: To compare the 12-month efficacy of 1 initial intravitreal injection of an anti-vascular endothelial growth factor (VEGF) agent followed by pro re nata (PRN) dosing with that of 3 initial monthly injections followed by PRN dosing in patients with macular edema (ME) after central retinal vein occlusion (CRVO). METHODS: Twenty-nine eyes received 1 initial injection (1+PRN group) and 20 received 3 monthly injections (3+PRN group). RESULTS: At month 12, changes in logMAR visual acuity from baseline were -0.172 ± 0.372 and -0.142 ± 0.317 in the 1+PRN and 3+PRN groups, respectively; the difference was not significant (p = 0.769). The number of anti-VEGF injections administered in the 3+PRN group (5.9 ± 2.1) was significantly greater than that in the 1+PRN group (4.1 ± 2.8; p = 0.022). CONCLUSION: When used for ME after CRVO, a 1+PRN regimen achieved 12-month outcomes similar to those of a 3+PRN regimen with fewer injections.

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.

ASSOCIATIONS BETWEEN MACULAR EDEMA AND CIRCULATORY STATUS IN EYES WITH RETINAL VEIN OCCLUSION: An Adaptive Optics Scanning Laser Ophthalmoscopy Study.

PURPOSE: To investigate associations between parafoveal microcirculatory status and foveal pathomorphology in eyes with macular edema (ME) secondary to retinal vein occlusion (RVO). METHODS: Ten consecutive patients (10 eyes) with acute retinal vein occlusion were enrolled, 9 eyes of which received intravitreal ranibizumab (IVR) injections. Foveal morphologic changes were examined via optical coherence tomography (OCT), and parafoveal circulatory status was assessed via adaptive optics scanning laser ophthalmoscopy (AO-SLO). RESULTS: The mean parafoveal aggregated erythrocyte velocity (AEV) measured by adaptive optics scanning laser ophthalmoscopy in eyes with retinal vein occlusion was 0.99 ± 0.43 mm/second at baseline, which was significantly lower than that of age-matched healthy subjects (1.41 ± 0.28 mm/second, P = 0.042). The longitudinal adaptive optics scanning laser ophthalmoscopy examinations of each patient showed that parafoveal AEV was strongly inversely correlated with optical coherence tomography-measured central foveal thickness (CFT) over the entire observation period. Using parafoveal AEV and central foveal thickness measurements obtained at the first and second examinations, we investigated associations between differences in parafoveal AEV and central foveal thickness, which were significantly and highly correlated (r = -0.84, P = 0.002). CONCLUSION: Using adaptive optics scanning laser ophthalmoscopy in eyes with retinal vein occlusion macular edema, we could quantitatively evaluate the parafoveal AEV. A reduction or an increase in parafoveal AEV may be a clinical marker for the resolution or development/progression of macular edema respectively.

ASSOCIATION BETWEEN RETINAL HEMORRHAGIC PATTERNS AND PERFUSION STATUS IN EYES WITH ACUTE CENTRAL RETINAL VEIN OCCLUSION.

PURPOSE: To evaluate peripheral retinal hemorrhagic patterns in eyes with acute central retinal vein occlusion, and to explore their clinical relevance in differentiating for the retinal perfusion status, through a prospective, and cross-sectional study. METHODS: Fifty eyes with acute central retinal vein occlusion were included. Retinal hemorrhagic patterns at the equator and retinal perfusion status were evaluated by ultra-wide field fundus photography and fluorescein angiography. RESULTS: Retinal perfusion was categorized as nonischemic in 29 eyes, ischemic in 18 eyes, and undeterminable in 3 eyes. None of the examined eyes had flame-shaped retinal hemorrhages in the periphery. All hemorrhages were rounded-dot or blot and were variable in size. Particle analysis was performed to quantify hemorrhage size, and showed higher values in eyes having larger blot hemorrhages, and lower values in eyes having dot or smaller blot hemorrhages. Mean size of maximum peripheral dot or blot hemorrhage was larger in eyes classified as ischemic (10,763.0 ± 5,946.3 pixels) than as nonischemic (2,839.9 ± 1,153.6 pixels, P < 0.001). The authors calculated area under the curve to investigate the ability of continuous variables to discriminate retinal perfusion status, which was 0.963 (P < 0.001) for mean size of maximum peripheral blot hemorrhages. CONCLUSION: The authors objectively evaluated retinal hemorrhagic patterns at the equator in eyes with acute central retinal vein occlusion using particle analysis. The resulting hemorrhage size measurement was considered to be often useful in determining retinal perfusion status. Because they can be noninvasively evaluated with readily available equipment, peripheral hemorrhagic patterns might be good clinical markers of retinal perfusion.

RANIBIZUMAB FOR MACULAR EDEMA AFTER BRANCH RETINAL VEIN OCCLUSION: One Initial Injection Versus Three Monthly Injections.

PURPOSE: To compare the 12-month-efficacy of 1 initial intravitreal ranibizumab injection (IVR) followed by pro re nata (PRN) dosing with that of three initial monthly IVR followed by PRN dosing in patients with macular edema (ME) after branch retinal vein occlusion. DESIGN: Prospective, interventional study. METHODS: Of 81 eyes, 42 received 1 initial IVR injection (1+PRN group) and 39 eyes received 3 monthly IVRs (3+PRN). Pro re nata injections were performed when fovea exudative changes were evident. RESULTS: At Month 12, the visual acuity (VA) changes from baseline were -0.245 ± 0.227 and -0.287 ± 0.222, in the 1+PRN and 3+PRN groups, respectively; there were no significant difference between groups (P = 0.728). The stratified analysis showed that patients with better VA (baseline VA >20/40) had similar significant improvement in VA at Month 12 (P < 0.001) to that of those with poorer VA (≤20/40). Better VA at Month 12 was significantly associated with younger age, better baseline VA, and thinner baseline central foveal thickness (P = 0.003, < 0.001, and < 0.001, respectively). Mean total number of IVR injections in the 1+PRN and 3+PRN groups were 3.8 ± 1.8 and 4.6 ± 1.4, respectively (P = 0.060). In both groups, shorter durations to the first PRN injection were associated with greater total PRN injection number (1+PRN, P = 0.006; 3+PRN; group, P < 0.001). CONCLUSION: In IVR treatment for ME after branch retinal vein occlusion, 1+PRN and 3+PRN regimens achieved similar 12-month functional outcomes. Patients with shorter durations to initial PRN injection may require more PRN treatments.

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.

Blood compatibility of poly(propylene glycol diester) and its water structure observed by differential scanning calorimetry and 2H-nuclear magnetic resonance spectroscopy.

Recently, we applied solution 2H-nuclear magnetic resonance spectroscopy (2H NMR) to analyze the water (deuterium oxide, D2O) structure in several biopolymers at ambient temperature. We established that polymers with good blood compatibility (i.e. poly(2-methoxyethyl acrylate) (PMEA)) have water observed at high magnetic fields (upfield) compared with bulk water. Polymers containing poly(propylene glycol) (PPG) or poly(propylene oxide) (PPO) exhibit good compatibility; however, the reason for this remains unclear. In addition, reports on the blood compatibility of PPO/PPG are limited. Therefore, PPG diester (PPGest) was prepared as a model polymer, and its blood compatibility and water structure were investigated. PPGest exhibited excellent blood compatibility. The water in PPGest was observed upfield by 2H NMR, and it was defined as non-freezing water via differential scanning calorimetry. Based on these observations, the relationship between the blood compatibility and water structure of PPGest is discussed by comparing with those of PMEA, and the reason for the good performance of PPG/PPO-based polymers is discussed.

AMP-activated protein kinase as a promoting factor, but complement and thrombin as limiting factors for acquisition of cytoprotection: implications for induction of accommodation.

Accommodation has been termed as a condition without graft rejection even in the presence of antidonor antibody. We previously reported an in vitro accommodation model, which demonstrated that preincubation of A/B antigen-expressing endothelial cells with anti-A/B antibody resulted in ERK inactivation followed by resistance to complement-mediated cytotoxicity through the induction of complement regulatory genes. However, under the in vivo condition, the effects of complement and coagulation system cannot be ignored. The purpose of this study is to find effective ways to navigate accommodation by exploring the relevant signal transduction. Preincubation with a low level of complement or thrombin failed to induce resistance to complement-mediated cytotoxicity. AMP-activated protein kinase (AMPK) activators such as resveratrol, AICAR and metformin protected endothelial cells against complement-mediated cytotoxicity through the increase in CD55, CD59, haem oxygenase-1 (HO-1) and ferritin heavy chain (ferritin H) genes, all of which were attenuated by AMPKα knock-down. Resveratrol counteracted the inhibitory effect of pretreated complement and thrombin on acquisition of resistance to complement-mediated cytotoxicity through AMPKα. AMPK regulation in endothelial cells could become the potential strategy to induce accommodation in clinical pro-inflammation and pro-coagulation.

Production of cloned pigs expressing human thrombomodulin in endothelial cells.

For long-term xenograft survival, coagulation control is one of the remaining critical issues. Our attention has been directed toward human thrombomodulin (hTM), because it is expected to exhibit the following beneficial effects on coagulation control and cytoprotection: (i) to solve the problem of molecular incompatibility in protein C activation; (ii) to exert a role as a physiological regulator, only when thrombin is formed; (iii) to suppress direct prothrombinase activity; and (iv) to have anti-inflammatory properties. hTM gene was transfected into pig (Landrace/Yorkshire) fibroblasts using pCAGGS expression vector and pPGK-puro vector. After puromycin selection, only fibroblasts expressing a high level of hTM were collected by cell sorting and then applied to nuclear transfer. Following electroactivation and subsequent culture, a total of 1547 cleaved embryos were transferred to seven surrogate mother pigs. Two healthy cloned piglets expressing hTM were born, successfully grew to maturity and produced normal progeny. Immunohistochemical staining of organs from F1 generation pigs demonstrated hTM expression in endothelial cells as well as parenchymal cells. High expression was observed particularly in endothelial cells of kidney and liver. Aortic endothelial cells from cloned pigs were found to express hTM levels similar to human umbilical vein endothelial cells (HUVEC) and to make it possible to convert protein C into activated protein C. The blockade of human endothelial cell protein C receptor (hEPCR) significantly reduced APC production in HUVEC, but not in hTM-PAEC. Although no bleeding tendency was observed in hTM-cloned pigs, activated partial thromboplastin time (APTT) was slightly prolonged and soluble hTM was detected in pig plasma. hTM was expressed in platelets and mononuclear cells, but not in RBC. Cloned pigs expressing hTM in endothelial cells at a comparable level to HUVEC were produced. As complete suppression of antigen-antibody reaction in the graft is essential for accurate assessment of transgene related to coagulation control, production of genetically engineered pigs expressing hTM and complement regulatory protein based on galactosyltransferase knockout is desired.

Comparative study on water structures of poly(tetrahydrofurfuryl acrylate) and poly(2-hydroxyethyl methacrylate) by nuclear magnetic resonance spectroscopy.

It is well known that poly(2-methoxyethyl acrylate) (PMEA) has good blood compatibility and its performance is attributed to its water structure. Recently, we applied solution nuclear magnetic resonance spectroscopy (solution-NMR) for analyzing the water structure in PMEA at ambient temperature and concluded that this method is useful because of the clear observation of the resonance peaks at low and high magnetic field (downfield and upfield, respectively) areas indicating the existence of more than two types of water. The present study was performed to compare the water structure of poly(tetrahydrofurfuryl acrylate) (PTHFA) and poly(2-hydroxyethyl methacrylate) (PHEMA) using solution 2H-NMR and deuterium oxide as water at the temperature range 15-45 °C. It was found that PTHFA has a different water structure from that of PHEMA. Water in PTHFA clearly showed two resonance peaks at downfield and upfield areas, with different spin-lattice relaxation times, T12H (high and low values, respectively). These observations are similar to those of PMEA. In contrast, PHEMA showed only one broad resonance peak (at downfield) with a low T12H value. Based on these observations, this study discusses the effect of water structures on the blood compatibility of these polymers.

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.

Significance of HLA class I antibody-induced antioxidant gene expression for endothelial cell protection against complement attack.

It has been observed that a graft organ continues to survive and function normally even in the presence of anti-graft antibodies. However, the mechanisms behind acquirement of this condition remain unknown. Here we report that the anti-HLA ligation on endothelial cells induces PI3K/AKT activation followed by antioxidant gene induction through Nrf2-mediated antioxidant-responsive element (ARE) activation. Activation of PI3K/AKT in endothelial cells by a low concentration of anti-HLA ligation enhances protection from complement attack. A real-time quantitative PCR and flow-cytometry experiment showed that ferritin H and HO-1 mRNAs were induced in a PI3K/AKT-dependent manner, while CD55 and CD59 expression were not enhanced by anti-HLA ligation. Anti-HLA ligation on endothelial cells activates ferritin H ARE and induces Nrf2 binding on its enhancer element. Finally, overexpression of Nrf2 in endothelial cells attenuates complement-mediated cytotoxicity. These experiments suggest that induction of PI3K/AKT-dependent cytoprotective genes by Nrf2 is an important mechanism to prevent complement attack. Thus, a protocol to activate this pathway would be a potential strategy for avoidance of graft rejection in transplantation.