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.

Role of multifunctional cell cycle modulators in advanced secondary hyperparathyroidism.

Long-term dialysis for patients with end stage renal disease leads to an unavoidable common complication, which is secondary hyperparathyroidism. Two histological patterns (nodular and diffuse hyperplasia) are detected, indicating that continuous uremia-related stimulation promotes parathyroid cell proliferation from diffuse to nodular growth. However, the key molecular mechanism is not fully understood, which narrows the range of therapeutic options for advanced secondary hyperparathyroidism. To address this issue, we utilized surgically resected normal and hyperplastic parathyroid glands to perform immunohistochemical analysis of a multifunctional cell cycle modulator, CCAAT enhancer binding protein (C/EBP)ß. In contrast to normal parathyroid tissue and diffuse hyperplasia, the intensity of C/EBPß staining was homogeneously increased in the parathyroid cells from nodules, along with a higher cyclin D1 labeling index (108.0 ± 19.5, mean ± SEM) and Ki-67 labeling index (31.70 ± 0.49). Normal and diffuse hyperplastic parathyroid glands had far fewer cyclin D1- and Ki-67-positive cells (P < 0.001). Immunofluorescent double staining showed abundant coexpression of Th235 (mitogen-activated protein kinase [MAPK] phosphorylation site) C/EBPß, along with upregulation of cytoplasmic Ras in nodular hyperplasia. In conclusion, hyperplastic parathyroid cells in nodules have an autonomous proliferation mechanism similar to that of cancer, in which C/EBPß is upregulated and phosphorylated to interact with the oncogenic Ras/MAPK pathway. C/EBPß may be a novel target molecule for blocking the growth circuit that underlies parathyroid tumorigenesis in secondary hyperparathyroidism.

A case report of recurrence of mixed cryoglobulinemic glomerulonephritis in a renal transplant recipient.

Recurrence of glomerulonephritis (GN) is one of the major risk factors of long-surviving renal graft dysfunction. Cryoglobulinemic glomerulonephritis of hepatitis-C virus (HCV)-negative patient is a rare cause of end-stage renal disease. There is little case report of recurrent cryoglobulinemic glomerulonephritis in negative HCV recipients after renal transplantation. We represent a renal allograft recipient of an interesting recurrent cryoglobulinemic glomerulonephritis. The patient was diagnosed with mixed cryoglobulinemic glomerulonephritis by kidney biopsy at the age of 32 . He had no HCV, HBV nor liver dysfunction. He received immunosuppressive therapy, however, was introduced to hemodialysis treatment after 13 yr. He received a cadaveric renal transplantation at the age of 50, and immunosuppressive treatment was started with ciclosporin, prednisolone and mycophenolate mofetil (MMF). Four yr after transplantation, he developed fever and purpura of lower limbs. His serum creatinine level did not increase, however, proteinuria, hematuria, hypocomplementemia, positive rheumatoid factor and mixed cryoglobulinemia were noted. Detailed analysis failed to reveal the composition of mixed cryoglobulinemia. The renal allograft biopsy showed membranoproliferative-type GN with monocyte and polynuclear leukocyte accumulation of capillary loops and small cellular crescent. Immunofluorescent study showed C3, IgG and IgM deposition of mesangial and capillary pattern. Regardless of steroid pulse therapy, hypocomplementemia and positive rheumatoid factor did not improve. Ten yr after transplantation, he was affected by cellulitis and sepsis. Afterward, rising of serum creatinine and nephrotic range proteinuria developed. The allograft biopsy revealed advanced cryoglobulinemic glomerulonephritis with characteristic vascular lesions. Electron microscopy showed organized subendothelial deposits compatible with cryoglobulinemic glomerulonephritis and proteinaceous thrombus in arteriole.

Removal of autografted parathyroid tissue for recurrent renal hyperparathyroidism in hemodialysis patients.

BACKGROUND: Recurrent renal hyperparathyroidism (HPT) is a serious problem after parathyroidectomy (PTx). We evaluated the frequency of graft-dependent recurrent HPT and the clinical outcomes after removal of the autograft. METHODS: Between March 1980 and January 2009, 2660 patients underwent total PTx with forearm autograft. After resection of all parathyroid glands, 30 pieces of 1 x 1 x 3 mm parathyroid tissue from diffuse hyperplasia, if possible, were autografted into brachioradial muscle. Graft-dependent recurrence of HPT was diagnosed by a high PTH gradient and detection of swollen autografts by palpation and/or MRI or US. RESULTS: In 248/2660 (9.3%) patients, removal of the graft was required a total of 327 times (53 patients required removal of the autograft several times). The cumulative frequency of graft-dependent recurrent HPT was 17.4% ten years after the initial PTx. Thirty-two patients underwent both resection of missed glands located in the neck or mediastinum and removal of the graft. En-bloc resection of autograft with surrounding muscle was required to avoid reoperation. When the intact PTH level dropped under 300 pg/ml, in the majority of patients renal HPT could be medically managed after the operation. The mean weight of the resected parathyroid tissue was 1583.7 mg. No specimen had histopathologically malignant features. Three patients suffered from hematoma in the wound. CONCLUSIONS: Graft-dependent recurrent renal HPT is not negligible. However, in the majority of patients, renal HPT can be controlled by removal of the autograft noninvasively. Total PTx with forearm autograft is preferable for hemodialysis patients, especially when long-term survival is expected.

Potential value of human thrombomodulin and DAF expression for coagulation control in pig-to-human xenotransplantation.

BACKGROUND: Problems of coagulation disorder remain to be resolved in pig-to-primate xenotransplantation. Molecular incompatibilities in the coagulation systems between pigs and humans, such as the thrombomodulin (TM)-protein C system or direct prothrombinase activity, have been suggested as possible causes. Coagulation and complement activation are closely related to each other. The purpose of this study was to elucidate the protective effects on the coagulation system of the expression of human TM and decay accelerating factor (hDAF) (for inhibition of complement activation) in pig endothelial cells. METHODS: Human aortic endothelial cells (HAEC), porcine aortic endothelial cells (PAEC), hDAF-expressing PAEC (hDAF-PAEC), hDAF/Endo-beta-galactosidase C-expressing PAEC (hDAF/EndoGalC-PAEC), hTM-expressing PAEC (hTM-PAEC), hDAF/hTM expressing-PAEC (hDAF/hTM-PAEC), and hDAF/EndoGalC/hTM-expressing PAEC (hDAF/EndoGalC/hTM-PAEC) were used in this study. Coagulation activity was examined by clotting, activated protein C (APC), and thrombin generation assay. RESULTS: A large difference was observed in clotting time of human plasma when exposed to PAEC (170 s) and HAEC (1020 s). hTM expression on PAEC was proven to produce a comparable level of APC to that produced by HAEC, which prolonged the clotting time, though not to the level of HAEC. Pretreatment with human sera considerably shortened the clotting time in PAEC (80 s). hDAF-PAEC significantly inhibited such a shortening of clotting time by reductions in tissue factor expression and thrombin generation. Thrombin generation through direct prothrombinase activity, which was detected only in PAEC, could be suppressed by hTM expression. Suppression of antibody binding and complement activation improved clotting time not in PAEC, but in PAEC expressing hTM. CONCLUSIONS: In addition to effective suppression of antibody-induced complement activation, hTM expression in PAEC may be essential for regulating procoagulant activity in xenotransplantation.

A case of acute rejection with adenovirus infection after ABO-incompatible kidney transplantation.

We report clinical and histopathologic findings of a case of acute rejection with adenovirus infection after kidney transplantation. A 63-yr-old woman with end-stage renal disease caused by lupus nephritis received an ABO-incompatible living kidney transplantation from her husband. On the 7th post-operative day (POD), she had fever, hematuria, and bladder irritation. Although she was treated with an antibiotic, the symptoms were not improved. We diagnosed adenovirus infection as positive with the urine shell vial method and blood PCR analysis. Cyclophosphamide was interrupted and immunoglobulin therapy was performed. However, urine output decreased and serum creatinine levels increased. An episode biopsy was performed on POD 20. We diagnosed acute antibody-mediated rejection. She was treated with plasma exchange for acute rejection and antiviral drug (rivabirin) for active adenovirus infection. However, the renal graft dysfunction was deemed irreversible and the renal graft was removed on POD 34. The graftectomy specimen showed acute rejection and acute tubular necrosis with adenovirus infection.

1,25-dihydroxyvitamin D synthesis after renal transplantation: the role of fibroblast growth factor 23 and cyclosporine.

1,25-dihydroxyvitamin D(3) (1,25D) is tightly regulated by circulating factors, containing fibroblast growth factor 23 (FGF23). However, this control is disturbed in chronic kidney disease. Renal transplantation (RTX) alters 1,25D homeostasis. To examine the clinical relevance of 1,25D in RTX, we drew blood samples from 27 renal transplant recipients (20 cyclosporine-based, seven non-cyclosporine-based) and examined serum concentrations of 25-hydroxyvitamin D(3) (25D), 1,25D, and FGF23. Our protocol for cyclosporine was as follows, an initial dose of 8 mg/kg two d before RTX, and subsequently adjusted on the basis of the pharmacokinetic profile. No baseline differences were observed between cyclosporine-based and non-cyclosporine-based regimens before RTX. All variables except 1,25D levels changed similarly between the two groups. In the cyclosporine-based regimen, 1,25D levels increased steeply on day 2 and re-increased from days 7 to 21. Post-transplant FGF23 levels sharply decreased until day 14. Interestingly, the cyclosporine-treated group revealed an unexpected tendency between circulating 1,25D and FGF23 on day 21. Multiple regression analyses indicated the cyclosporine pharmacokinetic profile as a significant predictor for 1,25D levels. Post-transplant 1,25D production is induced by a steep fall in serum FGF23 and prompt graft function on day 2; 1,25D levels thereafter may be stimulated by circulating abundant cyclosporine.

Stimulation index for PCNA mRNA in peripheral blood as immune function monitoring after renal transplantation.

Although more effective and potent immunosuppressive agents have recently reduced the incidence of acute rejection, drug-induced toxicity and infection caused by over-immunosuppression occasionally elicit a serious problem. However, no effective assay for evaluating overall patient's immune condition is in widespread use at present. We attempted to measure the stimulation index for mRNA of proliferating cell nuclear antigen (PCNA), which is synthesized in early G1 and S phases of the cell cycle and would be expected to reflect the proliferation capacity of T lymphocytes under the immunosuppressive condition. The stimulation index for PCNA mRNA seemed to be closely related to the immunosuppressive state of renal transplant recipients. Patients with stimulation index less than 2.0 tended to have viral reactivation after transplantation. It was suggested that PCNA mRNA monitoring in peripheral blood could provide a warning of possible over-immunosuppression as one simple assay for immune function monitoring.

Evaluation of interleukin-2 mRNA in whole blood as a parameter for monitoring cyclosporine pharmacodynamics.

Inhibition of cytokine production is the main immunosuppressive effect of cyclosporine (CsA), which is widely used in organ transplantation. Pharmacodynamic (PD) assay for evaluating the inhibition of interleukin-2 (IL-2) production for each patient could provide a more appropriate dosing regimen. We measured the suppression of IL-2 mRNA expression in whole blood following the addition of a range of CsA concentrations by a real-time reverse transcription-polymerase chain reaction (RT-PCR) method. Individual CsA sensitivity on the IL-2 mRNA expression was assessed with healthy subjects both in vitro and ex vivo. We also evaluated it in pre-transplant patients before taking immunosuppressive drugs. Sigmoid E(max) model was used to analyze the relationship between CsA concentration and IL-2 mRNA expression. The assay was completed within 8 h. The concentration that resulted in IC(50) showed high reproducibility and specificity among the healthy subjects (p<0.005, n=5). Ex vivo study indicated similar inhibition profiles to those of in vitro studies (n=3). The values of IC(50) obtained from patients (n=22) also showed large variations and were significantly lower than those from healthy subjects (p<0.05). Semi-quantitative RT-PCR was considered to be a rapid and reliable assay. Our data imply that measurement of IL-2 mRNA levels in whole blood could be valuable in monitoring CsA PD in transplant patients.

Restored cardiac conditions and left ventricular function after parathyroidectomy in a hemodialysis patient. Parathyroidectomy improves cardiac fatty acid metabolism assessed by 123I-BMIPP.

A 62 year-old female hemodialysis patient underwent parathyroidectomy to treat secondary hyperparathyroidism. On the preoperative assessment, the plasma levels of parathyroid hormone (PTH) and B-type natriuretic peptide (BNP) were elevated. Echocardiography showed reduced left ventricular (LV) contraction. Myocardial iodine-123-15-(p-iodophenyl)-3-(R,S) methylpentadecanoic acid ((123)I-BMIPP) scintigraphy showed moderately reduced tracer uptake in the postero-inferior area on single-photon emission computed tomography and decreased washout on the planar images. After parathyroidectomy, the plasma levels of PTH and BNP decreased, followed by improvement in LV contraction. Myocardial (123)I-BMIPP scintigraphy revealed that the washout on planar images had increased, which suggests that myocardial (123)I-BMIPP scintigraphy is useful for estimating the effect of parathyroidectomy on cardiac function.

Successful cross-breeding of cloned pigs expressing endo-beta-galactosidase C and human decay accelerating factor.

BACKGROUND: For successful organ xenotransplantation, genetically engineered pigs have been actively produced. Our attention has focused on (i) reduction of alphaGal expression by its digestion enzyme, endo-beta-galactosidase C (EndoGalC), and (ii) inhibition of complement activation by human decay accelerating factor (hDAF). Cell sorting and nuclear transfer enabled the effective production of cloned pigs expressing transgene at high levels. We report the successful cross-breeding of pigs expressing EndoGalC and hDAF. METHODS: After hDAF and EndoGalC genes were transfected into pig fibroblasts from the fetus of Landrace x Yorkshire and Meishan, respectively, transfected cells expressing transgenes effectively were collected using a cell sorter. Cloned pigs were produced using the technology of somatic cell nuclear transfer. After cross-breeding of cloned pigs, kidneys expressing both EndoGalC and hDAF were transplanted into baboons to examine the efficacy of gene transduction. RESULTS: Well-designed cloned pigs were produced by cross-breeding. alphaGal expression levels in cloned pigs were reduced up to 2 to 14%, compared to that in wild-type pigs. hDAF expression reached about 10- to 70-fold, compared to that in human umbilical vein endothelial cells. No congenital deformity was observed. There was no problem of increased stillbirth rate or growth retardation. Hyperacute rejection could be avoided in such a cloned pig to baboon kidney transplantation without any treatment for anti-pig antibody removal. However, grafts suffered from fibrin deposition as early as 1 h after transplantation, and were rejected after 1 week. CONCLUSIONS: Using a cell sorting system for effective collection of transfected cells, two types of cloned pigs were produced with a very high level of hDAF expression and a low level of alphaGal expression. Such genetic modification was effective in preventing hyperacute rejection, but there was an immediate lapse into procoagulation after transplantation, resulting in acute vascular rejection. Effective suppression of antibody binding to the graft would be necessary, even if a high level of hDAF is expressed.

Removal of blood group A/B antigen in organs by ex vivo and in vivo administration of endo-beta-galactosidase (ABase) for ABO-incompatible transplantation.

BACKGROUND: ABO incompatibility in organ transplantation is still a high risk factor for antibody-mediated rejection, despite the progress in effective treatments. We have explored the possibility of using the enzyme to remove the blood type A/B antigen in organs. METHODS: Recombinant endo-beta-galactosidase (ABase), which releases A/B antigen, was produced in E. coli BL-21. Human A/B red blood cells (RBC) were digested with ABase, and subjected to flow cytometric analysis after incubation with human sera. Purified recombinant ABase was intravenously administered to a baboon. Biopsies were taken from kidney and liver before and 1, 4 and 24 h after in vivo administration. Excised baboon kidneys were perfused with cold UW solution+/-purified recombinant ABase and preserved at 4 degrees C. Biopsies were taken before and 1 and 4 h after ex vivo perfusion. The change in A/B antigen expression was analyzed by immunohistochemical study. RESULTS: ABase removed 82% of A antigen and 95% of B antigen in human A/B red blood cells, and suppressed anti-A/B antibody binding and complement activation effectively. ABase was also found to remain active at 4 degrees C. In vivo infusion of ABase into a blood type A baboon demonstrated a marked reduction of A antigen expression in the glomeruli of kidney (85% at 1 h, 9% at 4 h and 13% at 24 h) and the sinusoids of liver (47% at 1 h, 1% at 4 h and 3% at 24 h) without serious adverse effects. After ex vivo perfusion and cold storage of excised baboon kidney (blood type B) with ABase, the expression levels of B antigen in glomeruli were reduced to 49% at 1 h and 6% at 4 h. CONCLUSIONS: This alternative approach might be useful for minimizing antibody removal and anti-B cell immunosuppression as an adjuvant therapy in ABO-incompatible kidney, liver and possibly heart transplantation.

Modified laparoscopic splenectomy: a beneficial technique for ABO-incompatible living donor renal transplantation candidates on hemodialysis.

Advances in laparoscopy have enabled minimally invasive surgical treatment of splenic diseases. Even with these advances, laparoscopic splenectomy in patients on dialysis can be difficult because of tissue fragility due to the underlying renal disease. We report a safe surgical technique for laparoscopic splenectomy in patients on maintenance dialysis that is suitable for use before ABO-incompatible living donor renal transplantation (LDRTx). Between June 1972 and December 2006, a total of 800 patients underwent LDRTx in our department, including 82 patients who underwent ABO-incompatible LDRTx. Between April 2001 and December 2006 we performed laparoscopic splenectomy in 48 hemodialysis patients as a pretreatment before ABO-incompatible LDRTx. Under general anesthesia the operation was performed using a new technique, referred to as the "splenic hilum lump method." We evaluated the surgical outcomes, such as the operative time, amount of blood loss, efficacy, and complications. The mean operative time was 131.6 +/- 38.4 min and mean blood loss was 126 +/- 395 mL. Blood transfusion was required in three patients. All cases had satisfactory kidney function after LDRTx and none developed kidney graft failure due to acute rejection. Almost all patients could walk the day after laparoscopic splenectomy and were satisfied with the cosmetic appearance of the scar after wound healing. The surgical technique we report here can be safely performed on patients with renal failure who require caution because of tissue fragility. Laparoscopic splenectomy is a safe, effective and less invasive operative procedure as a pretreatment for ABO-incompatible LDRTx.

Relationship between the dimension of parathyroid glands estimated by ultrasonography and the hyperplastic pattern in patients with renal hyperparathyroidism.

In renal hyperparathyroidism (HPT), the parathyroid glands initially proliferate diffusely and polyclonally, and are then transformed to monoclonal nodular hyperplasia with aggressive growth potential. In this study we evaluated the relationship between the maximal dimension of parathyroid glands estimated by ultrasonography (US) and the hyperplastic pattern of parathyroid glands in patients with renal HPT. Between October 1999 and December 2006, 141 patients who underwent total parathyroidectomy (PTx) with forearm autograft in our department were enrolled in this study. In these patients 308 parathyroid glands were detected by US before PTx. The largest dimension of the gland estimated preoperatively by US was correlated closely with its measurement at surgery (R2 was 0.31, P < 0.001). The maximal dimension of diffuse hyperplastic glands was significantly smaller than that of the glands with nodular hyperplastic glands (P < 0.001). There was a strong correlation between the pattern of parathyroid hyperplasia and the glandular diameter when we defined 8 mm as the maximal diameter estimated by US as a cut-off value. As a result of receiver operating characteristic analyses, using these criteria the US technique could predict nodular hyperplasia with a high sensitivity (78.9%) and specificity (78.7%). Parathyroid glands that are enlarged by more than 8 mm in the largest dimension estimated by US may represent glands with nodular hyperplasia.

Incidence of parathyroid glands located in thymus in patients with renal hyperparathyroidism.

BACKGROUND: Parathyroid glands are frequently located in thymus, and it is essential to resect thymic tissue from the neck incision, especially in surgery for renal hyperparathyroidism (HPT). METHODS: In this study, we evaluated the incidence, location, and type of intrathymic parathyroid glands in 902 patients who underwent initial parathyroidectomy (PTx) for advanced renal HPT in our department. Removal of the thymic tongues on both sides was routinely performed from the neck incision, and the thymic tissue was carefully examined both macroscopically and microscopically. RESULTS: Of the 902 patients in the study, 269 had only inferior parathyroid glands in the thymus, in 62 patients only supernumerary glands were found in the thymic tongue, and in 78 patients both inferior and supernumerary glands were present in thymic tissue. Therefore the incidence of patients with intrathymic glands was 45.3% (269 + 62 + 78 = 409/902). In 129 (92.1%) of 140 patients with supernumerary glands in the thymic tongue, these glands were detected only on histopathological examination, and about half of them were classified as the parathyromatosis type. CONCLUSIONS: In the human, parathyroid glands might be located in the thymus in about 50%. If the inferior gland/glands cannot be found around the inferior pole of thyroid lobe, it is very important to search for glands in the thymic tongue. Moreover, to avoid missing supernumerary glands, removal of the thymic tongue on both sides is essential in surgery for renal HPT.

Enzyme-linked immunosorbent assay for human leukocyte antigen antibody detection and urine protein test recommended for follow-up monitoring after renal transplantation.

BACKGROUND: Although the usefulness of posttransplant human leukocyte antigen (HLA) antibody monitoring has been demonstrated, detailed recommendations have not been worked out in its frequency, the type of patients and methods to be used. Enzyme-linked immunosorbent assay is a simple and cost-efficient assay. The urine protein test that reflects renal dysfunction is performed everywhere. We assessed the clinical value of HLA antibody and urine protein monitoring after renal transplantation. METHODS: Serum samples were consecutively collected from outpatients (n=323) in 2004 and in 2006. Because 18 had graft failure and 8 died with functioning graft for 2 years, 297 paired sera were tested for HLA antibody using enzyme-linked immunosorbent assay. Urine protein was determined to be positive when the dipstick protein reaction was+/-or over (20 mg/dL). RESULTS: Total 297 patients were divided according to the change of HLA antibody status. Only patients with all of (i) de novo HLA antibody production, (ii) continuous detection from peripheral blood, and (iii) positive urine protein test had a significantly higher serum creatinine than the others and demonstrated rapid deterioration of Cr (DeltaCr 1.26 mg/dL during 2 years). Negative change of HLA antibody stopped the increase of serum creatinine. CONCLUSION: The status of HLA antibody and urine protein provides useful information on graft prognosis. Although the tempo of graft injury is relatively slow, a yearly routine HLA antibody test for all patients and the attempt to reduce HLA antibody to negative levels is recommended, when HLA antibody is newly detected and urine protein test is positive.

Expression of parafibromin in distant metastatic parathyroid tumors in patients with advanced secondary hyperparathyroidism due to chronic kidney disease.

BACKGROUND: Recently, somatic inactivating mutations in HRPT2 have been reported in the majority of sporadic parathyroid carcinoma in primary hyperparathyroidism (HPT). Parafibromin is a tumor suppressor protein encoded by HRPT2, and loss of nuclear expression of parafibromin was found in approximately 70% of the carcinoma. In secondary HPT due to chronic kidney disease (CKD), parathyroid carcinoma is very rare and whether HRPT2 plays a role in the carcinogenesis in these cases is not clear. We evaluated the expression of parafibromin in hemodialysis patients with distant metastatic parathyroid tumors. METHODS: Between June 1973 and December 2006, 2,142 patients underwent parathyroidectomy (PTx) for secondary HPT in our department. We encountered five (0.23%) patients with distant metastatic parathyroid tumors. We evaluated the immunohistochemistry for parafibromin in eight primary parathyroid glands removed from the neck at the initial operation and/or at reoperation and seven distant metastatic tumors resected at reoperation. RESULTS: In only one lung metastatic parathyroid tumor, negative staining for parafibromin was detected. In the other three lung, two regional node, and one chest wall metastatic parathyroid tumor, parafibromin was strongly stained in the nuclei of the parathyroid cells. Among eight primary glands, except for one with weakly positive staining, the expression of parafibromin was detected diffusely and strongly. CONCLUSION: We conclude that the inactivating mutations and/or allelic loss of the HRPT2 gene may not play a major role in parathyroid carcinogenesis in secondary HPT due to CKD, but in these cases cancer development may be associated with a heterogeneous genetic disorder.

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.

Relation between human decay-accelerating factor (hDAF) expression in pig cells and inhibition of human serum anti-pig cytotoxicity: value of highly expressed hDAF for xenotransplantation.

BACKGROUND: Although the successful production of alpha1,3-galactosyltransferase-knockout (GT-KO) pigs has increased expectations of clinical xenotransplantation, additional modifications of genetically engineered pigs are still being explored, because even GT-KO pigs are incapable of inhibiting the host's immunological response completely. One of the potential candidates is a complement-regulatory protein, such as human decay-accelerating factor (hDAF). However, there are few reports on how high the expression level of hDAF in pig cells would be required for suppression of complement activation. The purpose of this study was to examine the relationship between the level of hDAF expression and its inhibitory effect on human serum cytotoxicity. METHODS: An expression (pCAGGS) vector containing the hDAF gene was transfected into pig fibroblasts using an electroporation system (Gene Pulser II). Forty-eight to fifty-two hours after transfection, the cells were stained with FITC-labeled anti-hDAF antibody and then applied to the cell sorter. hDAF-transfected cells with various expression levels were collected by gating on fluorescence intensity. The level of hDAF expression was determined relative to that in human control endothelial cells. Collected cells expressing x1, x5, x10, x15 and x30 hDAF were incubated into 96-well plates for 16 h, and the cells were subjected to 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) assay. RESULTS: hDAF expression levels in transfected cells at the time of MTT assay (16 h after sorting) were comparable to those immediately after sorting. hDAF expression in pig cells five times higher than in human endothelial cells was effective in inhibiting complement-dependent cytotoxicity of most human sera. However, 15- to 30-fold expression of hDAF was required for effective inhibition of human sera with the highest cytotoxic capacity. CONCLUSIONS: A much higher level of hDAF expression in pig cells than previously considered necessary might be required to provide additional benefit in inhibiting antibody-mediated rejection. Genetically engineered pigs that express very high levels of hDAF would be beneficial for xenotransplantation.