Virtual reality and augmented reality applications and simulation in vascular access management with three-dimensional visualization.

Obtaining adequate and precise anatomical information is mandatory to prevent vascular access-related complications in dialysis patients. For this purpose, we underwent Doppler ultrasound, vascular access angiogram, and plain computer-assisted tomography scan of the arm with vascular access. With the use of computer graphics software, the anatomical structure of the vascular access can be visualized three dimensionally which is shared among the staffs for precise and better recognition. Furthermore, created object is applicable for virtual reality and/or augmented reality presentation that provides useful means for education and practical procedures in the management of vascular access.

Prophylactic treatment of antibody-mediated rejection with high-dose mizoribine and pharmacokinetic study.

Although mizoribine (MZ), which inhibits inosine monophosphate dehydrogenase in the same way as mycophenolate mofetil, recently proved more effective when higher doses were administered than previously approved, neither the optimal dosage nor blood concentration has yet been clarified. We aimed at investigating the effect of high-doses of MZ on prevention of anti-donor antibody (Ab) production and acute Ab-mediated rejection (AMR) on the basis of the pharmacokinetic profile in a pig kidney transplantation model. Group 1 (n = 5) received cyclosporin microemulsion (6 mg/kg) and prednisolone (0.1 mg/kg). Groups 2, 3 and 4 (each n = 5) were treated, respectively, with 30, 10 and 3 mg/kg of MZ in addition to cyclosporin and prednisolone. The incidences of AMR in groups 1, 2, 3 and 4 were 5/5, 1/5, 3/5 and 5/5, respectively. Anti-donor IgG/IgM Ab levels (relative to pretransplantation levels) on day 14 in groups 1, 2, 3 and 4 were 10.3/9.3, 1.8/1.0, 2.3/1.8 and 6.5/3.5, respectively. While only 2 (28.6%) of seven pigs with Cmax > 3 microg/ml during the first 2 weeks had AMR, 7 (87.5%) of eight pigs with Cmax < 3 microg/ml elicited anti-donor Abs and experienced AMR (P = 0.0406). Effective Cmax seemed to be over 3 microg/ml at minimum. Higher doses of MZ efficiently prevented AMR. However, therapeutic drug monitoring is essential before clinical application.

Is absorption profile of cyclosporine really important for effective immunosuppression?

The clinical significance of cyclosporine (CsA) concentration 2 h postdose (C(2)) monitoring is widely recognized in organ transplantation, because C(2) value is considered to be a predictable surrogate marker of full area under the concentration-time curve (AUC), and/or a peak concentration value exhibits potent inhibition of calcineurin activity. However, the pharmacological advantage of absorption profile (AP) has not been fully elucidated. In a rat skin allotransplantation model, the authors evaluated the efficacy of AP by different dosage regimens (20, 25 or 30 mg/kg/d, once or twice daily) and routes (p.o. or i.v.), and examined whether high C(2) or AUC(0-4) is intrinsically valuable for effective immunosuppression. Graft survival was CsA dose-dependent and correlated with full AUC(0-24), rather than AP. The difference between the once and twice daily administrations did not influence full AUC(0-24) or immunosuppressive effect. Continuous intravenous infusion with flat pharmacokinetics also produced adequate immunosuppression as was observed in enteral administration at the same level of total exposure. The impact of high peak concentration in AP on immunosuppressive effect could not be found. It was suggested that AP would not have intrinsic pharmacodynamic value. However, absorption profiling was considered to be clinically useful in that C(2) value is a good surrogate marker of total exposure (AUC(0-24)).

Potential value of high-dose mizoribine as rescue therapy for ongoing acute humoral rejection.

Mizoribine (MZ) inhibits the proliferation of lymphocytes selectively via inhibition of inosine monophosphate dehydrogenase, like mycophenolate mofetil (MMF). The clinical dosage of MZ (2-5 mg/kg) is much lower than that of MMF (20-60 mg/kg). The purpose of this study was to examine whether high-dose MZ would be effective for treatment of acute humoral rejection. Renal transplantation was performed in a different pig strain combination. Group 1 (n = 2) received no treatment. Group 2 (n = 4) received cyclosporine microemulsion (6 mg/kg) and prednisolone (0.1 mg/kg) as baseline immunosuppression. Groups 3 (n = 4), 4 (n = 4) and 5 (n = 4) were additionally treated with MZ for rescue therapy, 30, 10 and 3 mg/kg, respectively, immediately after rejection was observed. All pigs developed acute vascular rejection between days 4 and 8. Complete reversal of acute rejection including reduction of elevated serum creatinine, suppression of anti-donor antibody production and pathological finding, was obtained in 3/4 (group 3), 1/4 (group 4) and 0/4 (group 5). Rescue with high-dose MZ (30 mg/kg) reversed ongoing acute humoral rejection. Such a high dose of MZ was tolerable for pigs. However, leukocytopenia was observed when MZ trough level was maintained over 10 mug/ml. Treatment with high-dose MZ would be applicable to a clinical trial, if blood level is carefully monitored.

Are N-glycolylneuraminic acid (Hanganutziu-Deicher) antigens important in pig-to-human xenotransplantation?

BACKGROUND: N-glycolylneuraminic acid (NeuGc) epitopes, so called Hanganutziu-Deicher (HD) antigens, which are widely expressed on endothelial cells of all mammals except humans, are considered to be potential targets for natural and elicited anti-nonGalalpha1-3 Gal (Gal) antibodies in humans. We previously reported that anti-NeuGc antibodies were not detected in healthy humans by enzyme-linked immunosorbent assay (ELISA) using NeuGc-GM3-coated plates, and no antibody production was observed in patients with a history of exposure to pig cells. However, a recent paper has revealed that (i) anti-NeuGc antibodies to porcine red blood cells (PRBC) are detectable in most healthy humans, and (ii) the majority of anti-nonGal antibodies are specific for NeuGc epitopes. The purpose of this study was to reassess whether NeuGc is important as an immunogenic nonGal epitope. METHODS: The binding of antibodies to PRBC and porcine aortic endothelial cells (PAEC) was compared. Cells were treated with (i) alpha-galactosidase, and then (ii) neuraminidase, which digests sialic acids, including NeuGc epitopes. Cells were incubated with human pooled sera, and applied to flow cytometric analysis. After enzyme digestion, almost complete reduction of Gal and NeuGc expression was confirmed by GS-IB4 and HU/Ch2-7 (a chicken monoclonal antibody against HD antigens), respectively. Trypsin, which removes membrane glycoproteins, and endoglycoceramidase which cleaves glycolipids, were used for differentiating between NeuGc-containing glycoproteins and glycolipids. RESULTS: Neuraminidase-treatment reduced the binding of immunoglobulin G (IgG) antibodies to PRBC; about half of the anti-nonGal IgG antibodies to PRBC were directed to NeuGc. In contrast, anti-nonGal antibodies to PAEC were not directed to NeuGc. Trypsin-treatment markedly reduced the expression of NeuGc only on PRBC. Endoglycoceramidase-treatment was followed by a greater reduction in NeuGc epitopes on PAEC than on PRBC. Most NeuGc on PRBC appeared to be linked to proteins, but most NeuGc on PAEC was expressed on glycolipids. CONCLUSIONS: Carbohydrate structures on PRBC are different from those on PAEC. Healthy human sera contain anti-nonGal IgG antibodies to NeuGc expressed on PRBC, but not on PAEC. We speculate that anti-nonGal IgG antibodies to PRBC can recognize both NeuGc and protein, and this may be the reason why such antibodies have not been detected by ELISA. A definite conclusion about the immunogenicity of NeuGc has not been obtained. More sera from patients (not from non-human primates) sensitized with porcine cells or organs need to be studied.

Amlodipine, but not MDR1 polymorphisms, alters the pharmacokinetics of cyclosporine A in Japanese kidney transplant recipients.

BACKGROUND: Cyclosporine A (CsA) is a critical immunosuppressive drug with a narrow therapeutic range and wide interindividual variation in its pharmacokinetics. Many factors, including P-glycoprotein (PGP), influence the oral bioavailability and interpatient variability of CsA. A number of polymorphisms have been identified in the human MDR1 gene, and some of them have been found to be associated with an altered expression of PGP. We have investigated the role of these polymorphisms in CsA absorption from kidney transplant recipients. In addition, we also investigated the effect of amlodipine on CsA absorption. METHODS: The area under the time-concentration curve from 0 to 2 hr (AUC(0-2)) estimated by the trapezoidal rule was used for the evaluation of extent of CsA absorption. The genotypes were identified by a polymerase chain reaction, restriction fragment length polymorphism analysis. RESULTS: No association was found between polymorphisms in the MDR1 and CsA AUC(0-2)/dose/kg. In contrast, the combination of amlodipine significantly increased CsA AUC(0-2)/dose/kg (706.2 microg x hr/L to 819.2 microg x hr/L, P<0.05). Furthermore, we attempted to compare MDR1 polymorphisms and the absorption of CsA again without patients receiving amlodipine, but there was still no significant difference. CONCLUSIONS: There is no relationship between polymorphisms for MDR1 and CsA absorption, suggesting polymorphisms for MDR1 cannot account for the interpatient variability of CsA. Amlodipine, which is the substrate of PGP, significantly increased CsA absorption. These results indicate that PGP plays a significant role in CsA absorption, but its polymorphisms could not influence the CsA absorption.

Reduction of alpha-galactosyl xenoantigen by expression of endo-beta-galactosidase C in pig endothelial cells.

BACKGROUND: Elimination of the Galalpha1-3Galbeta1-4GlcNAc (alphaGal) epitope has been considered to be essential for successful pig-to-human xenotransplantation but, unfortunately, has not been achieved. Endo-beta-galactosidase C (EndoGalC) is an endoglycosidase which cleaves the Galbeta1-4GlcNAc linkage in the alphaGal epitope and digests out the Galalpha1-3Gal disaccharide. Because of its potent activity in physiological pH conditions, EndoGalC can remove alphaGal epitopes expressed on the cell surface of pig erythrocytes and vascular endothelial cells almost completely. In vivo or ex vivo administration of EndoGalC successfully reduced alphaGal expression in pig kidneys to an undetectable level, but alphaGal epitopes soon reappeared. Gene expression of EndoGalC in pig cells was attempted to solve this problem. As the terminal alphaGal is transferred in the trans-Golgi network by alpha-1,3-galactosyltransferase (alpha1,3GT), colocalization of the EndoGalC gene with the alpha1,3GT gene was expected to be one of the most reliable ways to eliminate the alphaGal epitope. METHODS AND RESULTS: The sequence of pig alpha1,3GT, including the cytoplasmic tail, transmembrane domain and stem region, was ligated upstream of EndoGalC, and the conjugated gene was expressed in pig aortic endothelial cells and COS7 cells. Following the introduction of the gene, the alphaGal epitope on pig aortic endothelial cells was effectively reduced. Transfection studies in COS7 cells using EndoGalC combined with alpha1,3GT showed that the expressed EndoGalC was localized not only inside, but also outside, the cells. The expression of EndoGalC conjugated with a murine immunoglobulin (Igkappa)-chain signal sequence also showed a similar effect. CONCLUSIONS: These results suggest the effectiveness of gene transfer with EndoGalC into pig endothelial cells, and strongly encourage us to produce transgenic animals with the expressed enzyme.

Enzymatic removal of alphaGal antigen in pig kidneys by ex vivo and in vivo administration of endo-beta-galactosidase C.

Xenotransplantation using the pig as a donor species is considered to be a promising solution to the serious shortage of organ donors. Both hyperacute and acute vascular rejection (AVR) are believed to be associated with xenoreactive antibody binding to alphaGal epitopes on the pig vascular endothelial cells. Thus, suppression of this antigen-antibody reaction would appear essential for successful long-term xenograft survival. The purpose of this study was to examine the efficacy of ex vivo and in vivo administration of recombinant endo-beta-galactosidase C (EndoGalC which, in previous in vitro studies, has been proven to digest alphaGal antigens completely) on alphaGal epitopes expressed in pig kidneys. Excised pig kidneys were perfused with University of Wisconsin solution containing EndoGalC and preserved for 4 h. After cold storage, the pig kidney was transplanted into another pig. Ex vivo perfusion and cold storage with EndoGalC reduced alphaGal epitope expression on vascular endothelial cells to an undetectable level. However, alphaGal antigens began to be expressed again as early as 1 day after transplantation. The digestion of alphaGal epitopes by EndoGalC did not cause any damage to the kidney graft. EndoGalC was intravenously administered to two pigs (15 kg), without causing any serious adverse effect. Twelve hours later, >98% of alphaGal antigens on pig red blood cells (RBCs) had been digested. Immunohistochemical study revealed almost complete elimination of alphaGal expression on vascular endothelial cells of the kidney graft 4 and 8 h after in vivo administration, but reappearance within 24 h. EndoGalC was administered to a baboon after an interval of 2 months. The second administration did not result in any serious toxicity or reduction in efficacy. These results suggest that ex vivo and in vivo administration of EndoGalC is simple and useful in removing alphaGal epitopes from pig organs. As the effect of EndoGalC is temporary, multiple in vivo administrations of EndoGalC would be required to inhibit the reappearance of alphaGal epitopes. Alternatively, transgenic techniques of introducing the gene for EndoGalC into the donor organ might permanently prevent alphaGal expression.

Prevention of free-radical induced apoptosis by induction of human recombinant Cu, Zn-SOD in pig endothelial cells.

Vascular endothelial cells are the prime target in ischemia reperfusion injury. Growing evidence has shown that one of the main etiologies is considered to be reactive oxygen species (ROS) that induce endothelial-cell death either by necrosis or apoptosis. Cultured porcine endothelial cells were transfected with human copper, zinc-superoxide dismutase (h-Cu, Zn-SOD) to investigate whether these cells can prevent apoptosis from oxidative injury in vitro. The endothelial cells were cultured with SIN-1 (3-morpholinosydnonimine-N-ethylcarbanride) as a donor of peroxinitrite (ONOO(-)). The control cells without the gene transfection developed characteristic apoptotic changes both morphologically and biochemically when they were incubated with SIN-1 of 200 M. However, the cells showed necrosis predominantly when the concentration of SIN-1 was 1,000 M. On the other hand, the cells transfected with h-Cu, Zn-SOD showed significantly less evidence of apoptotic change after exposure to SIN-1. Nitric oxide (NO) did not significantly affect the viability of either the control cells or the transfected cells. One of the potent ROS, peroxinitrite, is considered to play a significant role in ischemia reperfusion injury. SIN-1 can produce peroxinitrite in vitro that induces endothelial-cell damage by apoptosis. This type of cytotoxicity can be successfully prevented by transfection of the h-Cu, Zn-SOD into the cells.