Functional analysis of platelet-derived growth factor receptor-ß in neural stem/progenitor cells.

Activation of neural stem/progenitor cells (NSPCs) is a potential therapeutic strategy of neurological disorders. In this study, NSPCs of subventricular zone were isolated and cultured from platelet-derived growth factor-ß-receptor-knockout (PDGFR-ß(-/-)) mice of postnatal day 1 (P1) and P28, and the roles of PDGFR-ß were examined in these cells. In PDGFR-ß-preserving control NSPCs, stem cell activities, such as numbers and diameters of secondary neurospheres, cell proliferation and survival rates, were significantly higher in P1 NSPCs than those in P28 NSPCs. In PDGFR-ß(-/-) NSPCs, most of these parameters were decreased as compared with age-matched controls. Among them, the decrease of secondary neurosphere formation was most striking in P1 and P28 PDGFR-ß(-/-) NSPCs and in P28 control NSPCs as compared with P1 control NSPCs. PCR-array and following quantitative real-time PCR (qRT-PCR) analyses demonstrated that expressions of fibroblast growth factor-2 (FGF2) and exons IV-IX of brain-derived neurotrophic factor (BDNF) were decreased, and noggin was increased in P1 PDGFR-ß(-/-) as compared with P1 controls. Addition of BDNF rescued the number and diameter of secondary neurospheres in P1 PDGFR-ß(-/-) NSPCs to similar levels as controls. The expressions of PDGFs and PDGFRs in control NSPCs were increased along with the differentiation-induction, where phosphorylated PDGFR-ß was co-localized with neuronal and astrocyte differentiation markers. In controls, the neuronal differentiation was decreased, and the glial differentiation was increased from P1 to P28 NSPCs. Compared with P1 controls, neuronal differentiation was reduced in P1 PDGFR-ß(-/-) NSPCs, whereas glial differentiation was comparable between the two genotypes. These results suggest that PDGFR-ß signaling is important for the self-renewal and multipotency of NSPCs, particularly in neonatal NSPCs. BDNF, FGF2, and noggin may be involved in the effects of PDGFR-ß signaling in these cells. Accordingly, the activation of PDGFR-ß in NSPCs may be a novel therapeutic strategy of neurological diseases.

[Thoracoscopic repair of diaphragmatic thinning for pleuroperitoneal communication; report of a case].

A 53-year-old man presented with massive right hydrothorax just after introduction of continuous ambulatory peritoneal dialysis (CAPD). Because the glucose concentration of pleural fluid was markedly high compared with that of serum, we diagnosed pleuroperitoneal communication. Thoracoscopic surgery was performed and thinning of the diaphragm was found. We sutured the diaphragm to repair the thin portion and performed pleurodesis with 50% glucose solution. He restarted CAPD 1 month post-operatively and continued at home without pleural effusion. Eight months post-operatively, he experienced dyspnea again and chest X-ray showed right hydrothorax. Although the cause of recurrent hydrothorax is unknown, it may be that not only surgical repair but also more intense pleurodesis is needed.

Esophagogastrostomy reconstruction after limited proximal gastrectomy.

BACKGROUND/AIMS: Recent advances in diagnostic techniques have led to the detection of an increasing number of early gastric cancers in the upper third of the stomach. The objective of this study was to determine the most appropriate surgical treatment for these cancers. METHODOLOGY: The clinicopathologic characteristics of 35 patients with early gastric cancer in the upper third of the stomach who underwent three different types of gastrectomies were reviewed retrospectively from hospital records between January 1992 and August 1999. RESULTS: Patients undergoing limited proximal gastrectomy with esophagogastrostomy reconstruction had shorter operation times and less blood loss than those for patients undergoing total gastrectomy or proximal gastrectomy with jejunal interposition. No lymph node metastasis was identified in any of these patients. Heartburn due to reflux esophagitis was seen in a few patients of each group, but they were successfully treated by antacids. The extreme reduction in food intake volume was more frequently experienced in patients with total gastrectomy than those with both proximal gastrectomies. When mortality due to other disease was excluded, all patients survived without recurrence. CONCLUSIONS: A limited proximal gastrectomy with esophagogastrostomy reconstruction decreased surgical risk and realized preservation of maximal function.

Synthesis and pharmacokinetics of a novel macromolecular prodrug of Tacrolimus (FK506), FK506-dextran conjugate.

A novel macromolecular prodrug of Tacrolimus (FK506), FK506-dextran conjugate, was developed and its physico-chemical, biological and pharmacokinetic characteristics were studied. The conjugate was estimated to contain 0.45% of FK506 and the coupling molar ratio was approximately 1:1 (dextran-FK-506). Adsorption experiments using ion exchangers indicated that FK506-dextran conjugate acted as a weakly negatively charged macromolecule. Low molecular weight radioactive compound(s), which was eluted in the same fractions as [(3)H]FK506, was released from [(3)H]FK506-dextran conjugate by chemical hydrolysis with a half-life of 150 h in phosphate buffer. In vitro immunosuppressive activity of the conjugate, as assessed by the rat lymphocyte stimulation test, was almost comparable to that of free FK506, suggesting that biologically active FK506 could be liberated from the conjugate. In vitro biodistribution studies demonstrated that conjugation with the dextran derivative dramatically changed the pharmacokinetic properties of FK506 after intravenous injection in rats. AUC of the FK506-dextran conjugate was almost 2000 times higher than that of free FK506 and organ uptake clearances of the conjugate were significantly smaller than those of the free drug. Thus, the present study has demonstrated that the FK506-dextran conjugate behaves as a prodrug of FK506 with an extended blood circulating time and can be expected to have an improved therapeutic potency.

[A case of cardiac rupture which occurred 34 years after mastectomy].

We experienced a case of cardiac rupture associated with mastectomy. A 78-year-old woman, who underwent left mastectomy 34 years before, complained of bleeding from a chest tumor located on the operative scar of the left mastectomy and postoperative irradiation. The tumor was noticed 10 months prior and became larger and repeated bleeding. It was first diagnosed as hemangioma by nearby practitioner. She was referred to the university hospital because of uncontrollable bleeding from the tumor. Without definite diagnosis, the tumor ruptured suddenly 52 days after the admission and the patient lapsed into the state of hemorrhagic shock and cardiopulmonary resuscitation was performed. Following emergency operation was performed on stand-by of cardiopulmonary bypass (CPB). The operation was successful with the aid of partial CPB and the final diagnosis of cardiac rupture was determined during the surgery. A case of cardiac rupture after mastectomy and radiation is rare, and this is the first report in the Japanese literature to date. The patient could be saved because the operation was performed on stand-by CPB.

Long-term prognosis of renal transplant surviving for over 10 yr, and clinical, renal and rehabilitation features of 20-yr successes.

Long-term mortality and morbidity was evaluated in 267 patients with a minimum follow-up of 10 yr and the physical status, graft function and quality of life in 15 patients with a functioning graft surviving for over 20 yr were reviewed. Actual patient and graft survival rates were 80.2% and 51.1% at 10 yr (n = 267) and 56.4% and 32.7% at 20 yr (n = 55), respectively. Although the rate of graft failure due to rejection was 4 times higher than that of patient death within 10 yr, it decreased to the level of patient death in the second decade. Dominant causes of death in patients with graft surviving for over 10 yr were hepatic failure due to viral hepatitis and malignancies. In 15 patients with graft currently surviving beyond 20 yr, while all patients have excellent graft function, malignancy occurred in 5, viral hepatitis in 3, aseptic necrosis in 3, and diabetes mellitus in one patient. No patient has suffered cardiovascular complications. Despite a high rate of morbidity, they show a satisfying status of rehabilitation (full time working 11/ 15, 73.3%). In order to attain more improved QOL in patients with long-term surviving renal transplant, close follow-up aiming at diminution of complications is required throughout the period after transplantation.

Immunological study of unresponsive state in rat hepatic transplant model. 1. Phenotypic and functional analyses of infiltrating cells.

In order to elucidate the immunological characteristics of rat liver transplantation, graft-infiltrating cells (GIC) isolated from rat hepatic allografts were analyzed phenotypically and functionally. GIC from long-surviving recipients (Brown Norway livers into Lewis hosts) and acutely rejecting recipients (DA livers into Lewis hosts) were compared. The relative proportions of all T cells and activated T cells determined by flow cytometry were significantly higher in acutely rejecting Lewis recipients than in long-surviving recipients on day 6 after grafting. Phenotypic kinesis of GIC on days 6, 14, and 45 after transplantation from long-surviving Lewis hosts was analyzed. Each proportion of all T cells, OX8-positive cells (cytotoxic T and natural killer cells), and OX39-positive cells (IL-2 receptor), was greatest on day 6 and decreased by day 45. Cytotoxic activity of GIC toward donor lymphocytes on day 6 was greater in acutely rejecting versus long-surviving recipients. These results demonstrate that an immunosuppressive mechanism is already present on day 6 posttransplantation, and that infiltration or activation of cytotoxic T cells is inhibited in the long-surviving rat hepatic allografts.

Immunological study of unresponsive state in rat hepatic transplant model. 2. Immunosuppressive factor in the serum from the tolerant hosts.

Our previous study demonstrated that Lewis (LEW) rat recipients engrafted with Brown-Norway (BN) rat liver displayed a long-term graft survival and that phenotypic and functional analyses of graft-infiltrating cells on day 6 postgrafting showed a lower proportion and activity of cytotoxic cells in long-term surviving hosts than LEW recipients engrafted with DA rat liver which showed acute rejection on day 9 postgrafting. In order to assess the immunological mechanisms of unresponsiveness, we analyzed the lymphocyte and serum from LEW recipients engrafted with BN liver. Spleen cells from tolerant LEW recipients on day 6 posttransplantation had no suppressor effect on the one-way mixed lymphocyte culture (MLC) reaction. On the other hand, when serum was added to MLC at a concentration of 6% of the total volume, it suppressed the mixed lymphocyte reaction (MLR) toward donor BN cells by 45.6%, but not toward third-party DA stimulator (-0.4%). Adoptive transfer of the serum from tolerant LEW hosts into the virgin secondary LEW hosts significantly prolonged the graft survival of BN kidneys from 7.8 +/- 0.2 to 14.7 +/- 1.6 days (p < 0.01), but not of third party DA kidney graft (mean survival time = 9.5 +/- 1.3 days). The in vitro study demonstrated that the suppressor factor in the serum inhibited the production of IL-2 as well as gamma-IFN in MLR. The suppressor factor was absorbed by LEW cells stimulated with BN cells in vitro, indicating that this factor was directed against recognition sites on responder T lymphocytes. These results showed that an antigen-specific tolerogenic factor which recognized the idiotype of the donor was released into the circulation through the process of BN liver grafting.

Induction of transplantation tolerance by chimeric donor/recipient class I RT1.Aa molecules.

Donor-specific transplantation tolerance was induced by administration of chimeric antigens in which four donor immunogenic amino acids (a.a.) were substituted onto the host class I MHC protein. We constructed chimeric rat RT1.Aa cDNA molecules by substituting nucleotides in the alpha1 helical region that encode 10 Lewis (LEW; RT1.A1) a.a., namely Asp58, Arg62, Glu63, Gln65, Lys66, Gly69, Asn70, Asn73, Ser77, and Asn80 ([alpha(1h)1]-RT1.Aa). The chimeric [alpha(1h)1]-RT1.Aa cDNA sequence was verified before transfection into Buffalo (BUF; RT1b) hepatoma cells. Interestingly, the helical regions of LEW rats (alpha(1h)1) and Wistar Furth (WF; RT1u) rats (alpha(1h)u) share four a.a. (Arg62, Glu63, Gln65, and Gly69). Consequently, subcutaneous administration of [alpha(1)1]-RT1.Aa transfectants (20x10(6); day -7) immunized BUF rats to reject in rapid fashion either LEW heart allografts (mean survival time [MST] = 4.2+/-0.4 days vs. 5.6+/-0.5 days in controls; P<0.001) or WF heart allografts (MST=4.4+/-0.6 days vs. 6.0+/-0.0 days in controls; P<0.002). Subcutaneous immunization of ACI (RT1a) rats with [a(1)1]-RT1.Aa transfectants (bearing 10 LEW donor a.a.) accelerated the rejection of LEW hearts (MST=5.0+/-0.8 days vs. 8.2+/-0.4 days in controls; P<0.001). In contrast, the same [a(1)1]-RT1.Aa transfectants (bearing only four WF donor a.a.) injected subcutaneously into ACI rats modestly prolonged the survival of WF hearts to 14.0+/-10.3 days from 5.4+/-0.5 days in controls (P<0.001). Furthermore, ACI recipients were rendered tolerant to WF heart allografts by a single injection via the portal vein of soluble [a(1)1]-RT1.Aa (but not RT1.Aa, RT1.Au, or [a(2)1]-RT1.Aa) antigens in conjunction with brief oral gavage treatment with cyclosporine. Thus, selected donor immunogenic a.a. (Arg62, Glu63, Gln65, and Gly69) of class I MHC antigens become tolerogenic when flanked by host sequences.

Synergistic interaction of 3 M KCl-extracted donor antigens (e-HAg) with cyclosporine or cyclosporine/sirolimus for prolongation of rat heart allograft survival.

Extracted donor histocompatibility antigens (e-HAg) may potentiate the effects of drugs to protect organ allografts from rejection. We examined the capacity of e-HAg when combined with cyclosporine (CsA) alone, sirolimus (rapamycin, RAPA) alone, or CsA/RAPA combinations to prolong heart allograft survival in rats. Wistar-Furth (WF; RT1u) rats that received CsA (10 mg/kg/day) by oral gavage for 3 (days 0, 1 and 2) or 7 (days 0, 1, 2, 3, 4, 5 and 6) consecutive days displayed modest prolongation of Brown Norway (BN; RT1n) heart allograft survival from a mean survival time of 7.2 +/- 0.8 days in untreated controls to 12.2 +/- 1.1 days and 18.6 +/- 2.7 days, respectively (p < 0.01). Although administration on the day of transplantation (day 0) of a single intravenous (i.v.) dose of BN e-HAg (5 mg/kg) failed to affect allograft survival, both three (days 0, 1 and 2) and five (days 0, 1, 2, 3 and 4) injections significantly potentiated the effect of a 3-day course of oral CsA (18.6 +/- 1.3 days (p < 0.01) and 20.0 +/- 1.4 days (p < 0.01), respectively) and of a 7-day course of oral CsA (25.3 +/- 4.4 days (p < 0.05) and 33.5 +/- 9.3 days (p < 0.01), respectively). Median-effect analysis confirmed a synergistic interaction between CsA (0.5 mg/kg x 7 days, i.v.) and e-HAg with combination index (CI) values less than 0.7 (CI = 1 shows additive interactions, CI < 1 synergistic, and CI > 1 antagonistic, interactions). In contrast, e-HAg failed to affect the immunosuppressive effect of RAPA. However, e-HAg (5.0 mg/kg x 3 days) significantly potentiated the effects of a 7-day or 14-day course of RAPA (0.01 mg/kg)/CsA (0.5 mg/kg) combination therapy, namely from 26.0 +/- 4.8 days with a 7-day treatment of CsA/RAPA alone to 32.6 +/- 3.6 days (p < 0.01) and from 28.2 +/- 2.7 days with a 14-day course of CsA/RAPA alone to 42.0 +/- 4.9 days (p < 0.05), respectively (CI = 0.2-0.5). Thus, e-HAg potentiates the immunosuppressive effects of CsA alone and of the CsA/RAPA combination, but not of sirolimus alone.