Xenoantigen, an αGal epitope-expression construct driven by the hTERT-promoter, specifically kills human pancreatic cancer cell line

BACKGROUND: We previously reported the usefulness of the αGal epitope as a target molecule for gene therapy against cancer. To induce cancer cell specific transcription of the αGal epitope, an expression vector which synthesizes the αGal epitope under the control of a promoter region of the human telomerase reverse transcriptase (hTERT), NK7, was constructed. METHODS: NK7 was transfected into a human pancreatic carcinoma cell line, MIA cells, and telomerase-negative SUSM-1 cells served controls. Expression of the αGal epitope was confirmed by flow cytometry using IB4 lectin. The susceptibility of transfected MIA cells to human natural antibodies, was examined using a complement-dependent cytotoxic cross-match test (CDC) and a flow cytometry using annexin V. RESULTS: The αGal epitope expression was detected only on the cell surfaces of NK7-transfected MIA cells, i.e., not on naive MIA cells or telomerase negative SUSM-1 cells. The CDC results indicated that MIA cells transfected with NK7 are susceptible to human natural antibody-mediated cell killing, and the differences, as compared to NK-7 transfected telomerase negative SUSM-1 cells or telomerase positive naïve MIA cells, were statistically significant. The flow cytometry using annexin V showed a higher number of the apoptotic cells in NK-7 transfected MIA cells than in naïve MIA cells. CONCLUSIONS: The results suggest that αGal epitope-expression, under the control of the hTERT-promoter, may be useful in cancer specific gene therapy

慢性期移植腎機能低下例におけるIV型コラーゲンの分布の変化に関する検討

IV型コラーゲンは基底膜の主要構成成分であり,生体内に広く分布している.移植後急性期腎機能低下例においては,腎臓のIV型コラーゲン分布に変化が認められる.今回我々は移植腎機能廃絶の主原因である慢性期移植腎機能低下症(chronic allograft nephropathy: CAN)におけるIV型コラーゲンの変化について検討した.当院において施行された腎移植患者を,移植腎機能に基づいて以下の群に分類し,組織所見について検討した.なおCAN症例は,光学顕微鏡所見で診断された症例を用いた.Group A(n=5):腎機能正常群,Group B(n=10):急性期移植腎機能低下群,Group C(n=35): CAN症例で血清クレアチニン値2～4mg/dl,Group D(n=15): CANで血清クレアチニン値>4mg/dl.各症例におけるIV型コラーゲン分布について,IV型コラーゲンのα1鎖に対するモノクローナル抗体であるJK199,JK132を用いて,蛍光顕微鏡により検討した.Group AではJK199,JK132ともにメサンギウム基質(MM),ボーマン嚢基底膜(BBM),尿細管基底膜(TBM)に対する反応のみを認めた.Group Bでは,JK199はMM,BBM,TBMに加え,糸球体基底膜(GBM)および腎間質(INS)に対する反応を認めた.JK132の反応性はGroup Aと同様であった.一方,Group CおよびGroup Dでは,JK199においてはMM,GBM,BBM,TBM,INSに対する反応性が増強していた.JK132についてはMM,BBM,TBMに加え,GBMおよびINSにおいても反応が認められた.また,JK199およびJK132の反応性はGroup CよりもGroup Dにおいて強い傾向が認められた.GBMおよびINSにおけるJK132の反応陽性化はCANに特異的な所見であることが示唆された.Previously, we demonstrated the altered formation of collagen IV, which is the main constituent of the basement membrane, in renal allografts by staining with two monoclonal antibodies against the α1 chain of collagen IV. In the present study, we investigated the alteration of collagen IV in chronic allograft nephropathy (CAN), which is an irreversible change that can occur in renal allografts. Biopsy specimens of normal kidneys (Group A: n=5) and acute rejection (Group B: n=10) were studied as controls. Fifty biopsy specimens from 41 patients who had been diagnosed as having CAN were divided into two groups, according to renal function: Group C (n=35), sCr 2～4 mg/dl, and Group D (n=15), sCr>4 mg/dl. Two monoclonal antibodies, JK199 and JK132, those recognize the α1 chain of collagen IV were used. In Group A, JK199 reacted with the glomerular basement membrane (GBM), the mesangial matrix (MM), the basement membrane of Bowman's capsule (BBM) and the tubular basement membrane (TBM). JK132 only reacted with the MM, BBM and TBM. In Group B, JK199 reacted with GBM, MM, BBM, TBM and the interstitium (INS). JK132 only reacted with MM, BBM and TBM. In Group C and Group D, JK199 and JK132 reacted universally with GBM and INS in addition to MM, BBM and TBM. The intensity of the reaction was higher in Group D than in Group C. Thus, the reactivity of JK132 with GBM and INS was a unique finding for the CAN specimens. These results suggest that collagen IV is upregulated in CAN and the reactivity of JK132 in GBM and INS may represent the point of irreversible dysfunction of renal allografts

慢性期移植腎機能低下例におけるIV型コラーゲンの分布の変化に関する検討

IV型コラーゲンは基底膜の主要構成成分であり,生体内に広く分布している.移植後急性期腎機能低下例においては,腎臓のIV型コラーゲン分布に変化が認められる.今回我々は移植腎機能廃絶の主原因である慢性期移植腎機能低下症(chronic allograft nephropathy: CAN)におけるIV型コラーゲンの変化について検討した.当院において施行された腎移植患者を,移植腎機能に基づいて以下の群に分類し,組織所見について検討した.なおCAN症例は,光学顕微鏡所見で診断された症例を用いた.Group A(n=5):腎機能正常群,Group B(n=10):急性期移植腎機能低下群,Group C(n=35): CAN症例で血清クレアチニン値2～4mg/dl,Group D(n=15): CANで血清クレアチニン値>4mg/dl.各症例におけるIV型コラーゲン分布について,IV型コラーゲンのα1鎖に対するモノクローナル抗体であるJK199,JK132を用いて,蛍光顕微鏡により検討した.Group AではJK199,JK132ともにメサンギウム基質(MM),ボーマン嚢基底膜(BBM),尿細管基底膜(TBM)に対する反応のみを認めた.Group Bでは,JK199はMM,BBM,TBMに加え,糸球体基底膜(GBM)および腎間質(INS)に対する反応を認めた.JK132の反応性はGroup Aと同様であった.一方,Group CおよびGroup Dでは,JK199においてはMM,GBM,BBM,TBM,INSに対する反応性が増強していた.JK132についてはMM,BBM,TBMに加え,GBMおよびINSにおいても反応が認められた.また,JK199およびJK132の反応性はGroup CよりもGroup Dにおいて強い傾向が認められた.GBMおよびINSにおけるJK132の反応陽性化はCANに特異的な所見であることが示唆された.Previously, we demonstrated the altered formation of collagen IV, which is the main constituent of the basement membrane, in renal allografts by staining with two monoclonal antibodies against the α1 chain of collagen IV. In the present study, we investigated the alteration of collagen IV in chronic allograft nephropathy (CAN), which is an irreversible change that can occur in renal allografts. Biopsy specimens of normal kidneys (Group A: n=5) and acute rejection (Group B: n=10) were studied as controls. Fifty biopsy specimens from 41 patients who had been diagnosed as having CAN were divided into two groups, according to renal function: Group C (n=35), sCr 2～4 mg/dl, and Group D (n=15), sCr>4 mg/dl. Two monoclonal antibodies, JK199 and JK132, those recognize the α1 chain of collagen IV were used. In Group A, JK199 reacted with the glomerular basement membrane (GBM), the mesangial matrix (MM), the basement membrane of Bowman\u27s capsule (BBM) and the tubular basement membrane (TBM). JK132 only reacted with the MM, BBM and TBM. In Group B, JK199 reacted with GBM, MM, BBM, TBM and the interstitium (INS). JK132 only reacted with MM, BBM and TBM. In Group C and Group D, JK199 and JK132 reacted universally with GBM and INS in addition to MM, BBM and TBM. The intensity of the reaction was higher in Group D than in Group C. Thus, the reactivity of JK132 with GBM and INS was a unique finding for the CAN specimens. These results suggest that collagen IV is upregulated in CAN and the reactivity of JK132 in GBM and INS may represent the point of irreversible dysfunction of renal allografts

ラット小腸の虚血再酸素化傷害に対するアスコルビン酸の抑制効果について

様々の臓器における虚血後再酸素化(ischemia/reoxygenation; I/R)時の細胞や機能の傷害は,発生する種々のフリーラジカルや,過酸化脂質によると考えられている.一方,アスコルビン酸(AsA)は,低濃度では過酸化を促進し,高濃度では逆に抑制することが知られている.ラット小腸においてI/R時の傷害の程度とAsAによる傷害抑制効果について生化学的,病理学的に検討した.AsAとグルタチオン(GSH)を,雄性Wistar系ラットに腹腔内投与し,60分後開腹,上腸間膜動脈を遮断し,全小腸を60分間虚血状態とした.再酸素化後20分で,全小腸を摘出して検索し,以下の結果をえた.AsA投与群では,過酸化脂質の産生は,有意に抑制され(LOOH Control: 26.30±1.98, AsA: 16.75±1.43 nmol/g tissue, p<0.01),また,glutaminase活性は,再酸素化後も有意に高く保たれ(Control: 0.41±0.04, AsA: 1.02±0.05μmol glutamate/h/mg tissue, p<0.05),組織傷害も軽微であった.また,還元型GSHレベルは,再酸素化後も高値に保たれた(Control:0.22±0.02, AsA:1.34±0.12μmol/g tissue, p<0.01).AsAは,ラット小腸において抗酸化剤として作用し,I/R傷害を軽減させることが明らかとなった.Ischemia/reoxygenation (I/R) injury is observed in various organs, where some free radicals and lipid peroxides are believed to play an important role. We examined the preventive effects of ascorbic acid (AsA), one of antioxidant agents, on the I/R injury of the rat small intestine. AsA or glutathione was intraperitoneally administered to male Wistar rats weighing 250～300 g after one-day fasting. The entire small intestine was resected at three different points, just before ischemia, after ischemia, and after 20 min of reoxygenation. The specimens were used for the assays of lipid peroxides, glutathione, and glutaminase activity and for histological examination. In AsA-treated group, the production of lipid peroxides after reoxygenation was significantly suppressed and the ratio of reduced form of glutathione to the total glutathione was also significantly high. Tissue glutaminase activity decreased to less extent, and the degree of injury by reoxygenation was apparently less marked in this AsA-treated group. AsA works as an antioxidant against the peroxidative tissue injury possibly by scavenging free radicals and reducing the peroxidative reaction. The effects of AsA are expected to be very useful in small bowel transplantation and various surgical procedures

ラット小腸の虚血再酸素化傷害に対するアスコルビン酸の抑制効果について

様々の臓器における虚血後再酸素化(ischemia/reoxygenation; I/R)時の細胞や機能の傷害は,発生する種々のフリーラジカルや,過酸化脂質によると考えられている.一方,アスコルビン酸(AsA)は,低濃度では過酸化を促進し,高濃度では逆に抑制することが知られている.ラット小腸においてI/R時の傷害の程度とAsAによる傷害抑制効果について生化学的,病理学的に検討した.AsAとグルタチオン(GSH)を,雄性Wistar系ラットに腹腔内投与し,60分後開腹,上腸間膜動脈を遮断し,全小腸を60分間虚血状態とした.再酸素化後20分で,全小腸を摘出して検索し,以下の結果をえた.AsA投与群では,過酸化脂質の産生は,有意に抑制され(LOOH Control: 26.30±1.98, AsA: 16.75±1.43 nmol/g tissue, p<0.01),また,glutaminase活性は,再酸素化後も有意に高く保たれ(Control: 0.41±0.04, AsA: 1.02±0.05μmol glutamate/h/mg tissue, p<0.05),組織傷害も軽微であった.また,還元型GSHレベルは,再酸素化後も高値に保たれた(Control:0.22±0.02, AsA:1.34±0.12μmol/g tissue, p<0.01).AsAは,ラット小腸において抗酸化剤として作用し,I/R傷害を軽減させることが明らかとなった.Ischemia/reoxygenation (I/R) injury is observed in various organs, where some free radicals and lipid peroxides are believed to play an important role. We examined the preventive effects of ascorbic acid (AsA), one of antioxidant agents, on the I/R injury of the rat small intestine. AsA or glutathione was intraperitoneally administered to male Wistar rats weighing 250～300 g after one-day fasting. The entire small intestine was resected at three different points, just before ischemia, after ischemia, and after 20 min of reoxygenation. The specimens were used for the assays of lipid peroxides, glutathione, and glutaminase activity and for histological examination. In AsA-treated group, the production of lipid peroxides after reoxygenation was significantly suppressed and the ratio of reduced form of glutathione to the total glutathione was also significantly high. Tissue glutaminase activity decreased to less extent, and the degree of injury by reoxygenation was apparently less marked in this AsA-treated group. AsA works as an antioxidant against the peroxidative tissue injury possibly by scavenging free radicals and reducing the peroxidative reaction. The effects of AsA are expected to be very useful in small bowel transplantation and various surgical procedures

Preoperative assessment system for hand-assisted laparoscopic donor nephrectomy by discriminant analysis.

We developed a preoperative assessment system to predict surgical workload in hand-assisted laparoscopic donor nephrectomy (HALDNx) using the normal-based linear discriminant rule (NLDR). A total of 128 cases of left HALDNx performed by a single operator were used as training data. Surgical workload was measured by operative time. The optimized model had 9 explanatory variables: age, total protein, total cholesterol, number of renal arteries (numberRA), 4 variables of perinephric fat (PNF), and thickness of subcutaneous fat. This model was validated using cross-validation and the .632 estimator to estimate discrimination rates with future test data. PNF and numberRA were the predominant factors affecting workload followed by the computed tomography value of PNF, body weight, and male sex. The estimated accuracy of the prediction system was 94.6%. The complication rate was 9.38% and did not correlate with surgical workload. We also made our program available online for constructing assessment functions from other cohort data. In conclusion, the surgical workload of HALDNx could be predicted with PNF and numberRA as the dominant risk factors

Preoperative Low-Density Lipoprotein Apheresis for Preventing Recurrence of Focal Segmental Glomerulosclerosis after Kidney Transplantation

Background. Focal segmental glomerulosclerosis (FSGS) often develops rapidly and frequently progresses to renal failure, while the recurrence rate after kidney transplantation is 20–50%. We performed low-density lipoprotein (LDL) apheresis before kidney transplantation in FSGS patients to prevent recurrence. Methods. Five adult patients with chronic renal failure due to FSGS undergoing living related donor kidney transplantation were investigated retrospectively. LDL apheresis was done 1-2 times before transplantation. Postoperative renal function and recurrence of FSGS were assessed. Results. The patients were two men and three women aged 24 to 41 years. The observation period ranged from 60 days to 22 months. Preoperative LDL apheresis was performed once in one patient and twice in four patients. Blood LDL cholesterol levels were normal before LDL apheresis and remained normal both after LDL apheresis and after kidney transplantation. Additional LDL apheresis was performed once in one patient with mild proteinuria after transplantation. The renal graft survived in all patients and there was no evidence of recurrent FSGS. Conclusions. Although the observation period was short, FSGS did not recur in all 5 patients receiving preoperative LDL apheresis. These results suggest that LDL apheresis can be effective in preventing recurrence of FSGS after kidney transplantation

Duodenal Graft Perforation after Simultaneous Pancreas-Kidney Transplantation

A 45-year-old woman with type 1 diabetes and chronic renal failure on dialysis underwent simultaneous pancreas-kidney transplantation from a brain dead donor. On postoperative day 15, acute generalized peritonitis was diagnosed and emergency laparotomy was performed. Perforation of the donor duodenum was found, which had apparently resulted from duodenal compression by the tip of the intestinal fistula tube placed for decompression. The perforation was sutured and the intestinal fistula tube was exchanged. Following this, perforation repeatedly recurred at the same site and open repair at laparotomy was required a total of four times. The fourth operation involved both suturing the perforation and covering it with ileum, after which there was no further recurrence. The patient was discharged on posttransplantation day 219, with the pancreas and kidney grafts both functioning well. This report presents a rare complication of simultaneous pancreas-kidney transplantation