Expression of cell cycle regulators during smooth muscle cell proliferation after balloon catheter injury of rat artery

Intimal hyperplasia is defined as the abnormal migration and proliferation of vascular smooth muscle cells (VSMCs) with deposition of extracellular matrix. However, the cell cycle regulatory mechanisms of injury-induced VSMC proliferation are largely unknown. To examine the expression kinetics of cell cycle regulatory factors which is known to be worked positively or negatively, we used rat balloon injury model. Marked induction of proliferating cell nuclear antigen (PCNA), G1/S cyclin-dependent kinase (cdk2), and its regulatory subunit (cyclin E) occurred between 1 and 3 days after balloon arterial injury, and this was sustained for up to 7 days and then declined. However, the induction of the negative regulators, p21 and p27, occurred between 3 and 5 days of injury, peaked after 7 and 14 days and was then sustained. VSMC proliferation after balloon catheter injury of the rat iliac artery is associated with coordinated expression of positive (cdk2, cyclin E and PCNA) and negative (p21, p27) regulators. Cell cycle regulators such as cdk2, cyclin E, p21, p27 may be suitable targets for the control of intimal hyperplasia.     

Veno-occlusive disease (VOD) of the liver in Korean patients following allogeneic bone marrow transplantation (BMT): efficacy of recombinant human tissue plasminogen activator (rt-PA) treatment.

Veno-occlusive disease (VOD) of the liver is a clinical syndrome characterized by hyperbilirubinemia, painful hepatomegaly, and fluid retention. In the bone marrow transplantation (BMT) setting, VOD is caused by dose-intensive chemotherapy and/or radiotherapy used to prepare patients for transplant. VOD occurs in up to 50% of the patients who undergo BMT and is usually associated with a high mortality rate. Until recently, there was no proven effective medical therapy for this condition once it was clinically apparent. We report here on the frequency and treatment result of VOD with rt-PA in our allogeneic BMT patients. Eight patients (median age 28.5 years) underwent allogeneic BMT from December, 1993 to June, 1995 in Asan Medical Center. Six leukemia patients were prepared for BMT with busulfan and cyclophosphmide, while two aplastic anemia patients received cyclophosphamide and antithymocyte globulin. VOD was defined as having two of the following features before day 20 posttransplant: jaundice (bilirubin > or = 2 mg/dL), tender hepatomegaly and/or right upper quadrant pain, ascites and/or unexplained weight gain (> 2% from baseline). All patients who were diagnosed with VOD received rt-PA (10-20 mg/day) and heparin (10,000 U/day). Three (37.5%) of the eight patients developed VOD that occurred between 6 and 10 days posttransplant. All three patients developed jaundice, weight gain, and tender hepatomegaly. Ascites and renal insufficiency occurred in two patients and pleural effusion in one patient. rt-PA and heparin were begun 6 to 26 days posttransplant and rt-PA was administered for 7 to 14 days. All three patients responded to the therapy; bilirubin levels began to decrease at 4 to 13 days from the start of therapy. They are all alive at day 111, 316, and 548 days posttransplant. None of the patients had significant hemorrhagic complications after rt-PA treatment. Prolonged administration of rt-PA was feasible without bleeding episode and it seems that rt-PA may alter the natural course of VOD.     

Pharmacokinetics of theophylline and caffeine after intravenous administration of aminophylline to premature neonates in Korea

Theophylline has been widely used to treat apnea of premature neonates. The purpose of this study was to compare the pharmacokinetic parameters of theophylline and caffeine after intravenous administration of aminophylline to seven Korean low-birthweight neonates with apnea to those in other countries. The serum concentrations of theophylline and caffeine were measured simultaneously by high-performance liquid-chromatography (HPLC). The mean (+/- S.E.M.) birth weight and gestational period were 1190 +/- 253 g and 31.5 +/- 1.99 weeks, respectively. The mean (+/- S.E.M.) theophylline maintenance dosage was 1.28 +/- 0.15 mg/kg (given as equivalent aminophylline solution) every six hours. The mean (+/- S.E.M.) volume of distribution, 0.937 +/- 0.232 l/kg, elimination rate constant, 0.0249 +/- 0.0095/h, elimination half-life, 32.1 +/- 12.1 h, and total body clearance, 21.7 +/- 6.18 ml/h/kg, of theophylline in Korean premature neonates were comparable to the values of neonates in other countries. For caffeine, the mean (+/- S.E.M.) elimination half-life was 95.1 +/- 25.4 h and the elimination rate constant was 0.0079 +/- 0.0024/h. The mean (+/- S.E.M.) serum concentrations of theophylline and caffeine on the sixth day after aminophylline infusion were 10.4 +/- 2.28 microg/ml (range, 6.38-13.4 microg/ml) and 2.94 +/- 0.98 microg/ml (range, 1.80-4.44 microg/ml), respectively. The mean (+/- S.E.M.) caffeine to theophylline concentration ratio on the day after discontinuation of aminophylline infusion was 0.71 +/- 0.23 (range, 0.39-1.03).     

A pilot study of bortezomib in Korean patients with relapsed or refractory myeloma

Recent clinical trials showed that bortezomib, a novel proteasome inhibitor, had therapeutic activity in multiple myeloma. However, there was no data about the feasibility of bortezomib in Korean patients. We performed a pilot study of bortezomib in patients with relapsed or refractory myeloma (1.3 mg/m2 twice weekly for 2 week in a 3-week cycle). Seven patients were enrolled. The median age of patients was 59 yr. All patients previously received VAD (vincristine, doxorubicin and dexamethasone) and thalidomide chemotherapy. Three patients previously received alkylator-containing chemotherapy and 4 patients, autologous stem cell transplantation. Bortezomib monotherapy resulted in 3 partial remissions (43%), 3 no changes (43%) and 1 progressive disease (14%). One patient who had no response to bortezomib monotherapy experienced partial remission after addition of dexamethasone to bortezomib. The most common serious toxicity was thrombocytopenia (grade 3/4, 10 of 20 cycles (50%)) and grade 3 peripheral neuropathy was developed in 2 of 20 cycles (10%). Drug-related adverse event led to discontinuation of bortezomib in 1 patient. There was no treatment related mortality. Overall, bortezomib seems to be effective and feasible. Conduction of larger clinical studies on Korean patients is necessary to characterize clinical efficacy and safety of bortezomib more precisely.     

BK polyomavirus interstitial nephritis in a renal allograft recipient

Human polyomavirus (PV) interstitial nephritis has recently been recognized as a cause of severe renal allograft dysfunction. It occurs in immunosuppressed patients after reactivation of the latent virus PV type BK (BK virus) in the renal epithelium. BK disease is defined as a morphologically manifest renal infection with cytopathic signs accompanied by varying degrees of interstitial inflammatory cell infiltrates and functional impairment. It is also identified by the presence of cells containing viral inclusion bodies (decoy cells) in the urine. The authors report a case of BK PV interstitial nephritis in a 36-year-old renal allograft recipient. Under light microscopy the chief diagnostic indicator was detection of intranuclear viral inclusions, which were found exclusively in tubular epithelial cells. Cells with viral changes were often enlarged with nuclear atypia and chromatin basophilia. Widespread interstitial plasma cell infiltrates associated with tubulitis were present. Intranuclear paracrystalline arrays of virus particles 35-38 nm in diameter were present as characteristic ultrastructural indicators. Urine samples revealed decoy cells with ground-glass-type intranuclear inclusions positive for BK virus by electron microcopy.     

Lymphocyte subpopulations. Human red blood cell rosettes.

Human red blood cells (HRBC) even without prior neuraminidase treatment, could form rosettes with human peripheral blood lymphocytes in vitro. The optimum conditions for forming these rosettes were a pH of 7-0 and a medium with 5% bovine serum albumin (BSA). Rosette proportions became much less at a different pH or using lower concentrations of BSA, or replacing BSA with foetal calf sera (FCS) or human sera. Rosette formation was also promoted by prior treatment of HRBC or lymphocytes with neuraminidase. Mixed rosettes of HRBC and sheep red blood cells (SRBC) showed that HRBC receptors were detectable only on lymphocytes that possessed SRBC receptors, suggesting that HRBC rosette-forming cells were probably thymus-derived (T) cells. Next, the properties of human red blood cell (HRBC) and sheep red blood cell (SRBC) rosette-forming cells were investigated by comparing the ability of human peripheral blood lymphocytes to form these two types of rosettes after treatment with various inhibitory reagents. HRBC rosettes were relatively more resistant to inhibition with: (1) proteolytic agents, such as trypsin, alpha-chymotrypsin and pronase; (2) anti-thymocyte serum (ATS); (3) metabolic inhibitors, such as sodium azide and 2,4-dinitrophenol (DNP); (4) cytochalasin B. On further incubation after trypsinization, the lymphocytes recovered some ability to form SRBC rosettes, but continued to lose more of their capability to form HRBC rosettes. All these results were regarded as circumstantial evidence that the HRBC rosettes might represent a subpopulation of human T lymphocytes.     

Replacement of a tracheal defect with autogenous mucosa lined tracheal prosthesis made from polypropylene mesh

Reliable prosthetic or tissue grafts for the trachea have not, as yet, been developed for reconstruction of large, circumferential tracheal defects. Major limitations are anastomotic dehiscence and stenosis, attributed to the poor epithelialization and vascularization of the prosthetic graft. We have developed a new tracheal prosthesis that has a well vascularized and viable mucosa. The prosthesis consists of a Prolene mesh reinforced with polypropylene rings, and coated with gelatin. We lined the luminal surface of the prosthesis with transplanted autogenous oral mucosa, wrapped the prosthesis with greater omentum, and placed it in the peritoneal cavity for 2 weeks. Complete surgical resection and replacement of a segment (5 cm in length, 8 to 10 tracheal rings) of the thoracic trachea was then performed in nine adult mongrel dogs. The transplanted mucosa was well vascularized and maintained its normal histology in prereplacement analysis. Dogs with tracheal replacement regained their full activity and did not show any respiratory problems until sacrifice at 1, 2, and 6 months. After 6 months, the prostheses were completely incorporated by the host trachea in all dogs and confluent epithelialization was confirmed histologically from the upper to the lower anastomotic site of the prosthesis; furthermore, the transplanted mucosal cells had changed to ciliated columnar epithelium.     

Background nonselective cationic current and the resting membrane potential in rabbit aorta endothelial cells

The ion channel conductances that regulate the membrane potential was investigated by using a perforated patch-clamp technique in rabbit aorta endothelial cells (RAECs). The whole-cell current/voltage (I-V) relation showed a slight outward rectification under physiological ionic conditions. The resting membrane potential was -23.3 +/- 1.1 mV (mean +/- SEM, n = 19). The slope conductances at the potentials of -80 and 50 mV were 31.0 +/- 4.0 and 62.8 +/- 7.1 pS pF(-1), respectively (n = 15). Changes in the extracellular and intracellular Cl(-) concentrations did not affect the reversal potential on I-V curves. The background nonselective cationic (NSC) current was isolated after the K(+) current was suppressed. The relative permeabilities calculated from the changes in reversal potentials using the constant-field theory were P(K):P(Cs):P(Na):P(Li) = 1:0.87:0.40:0.27 and P(Cs):P(Ca) = 1:0.21. Increases in the external Ca(2+) decreased the background NSC current in a dose-dependent manner. The concentration for half block by Ca(2+) was 1.1 +/- 0.3 mM (n = 7). Through the continuous recording of the membrane potential in a current-clamp mode, it was found that the background NSC conductance is the major determinant of resting membrane potential. Taken together, it could be concluded that the background NSC channels function as the major determinant for the resting membrane potential and can be responsible for the background Ca(2+) entry pathway in freshly isolated RAECs.     

Sustained release of ascorbate-2-phosphate and dexamethasone from porous PLGA scaffolds for bone tissue engineering using mesenchymal stem cells

The purpose of this research was to develop porous poly(D,L-lactide-co-glycolide) (PLGA) scaffolds from which ascorbate-2-phosphate (AsAP) and dexamethasone (Dex) are continuously released for a month for osteogenesis of mesenchymal stem cells for bone tissue engineering. Porous PLGA matrices containing AsAP and Dex were prepared by solvent casting/particulate leaching method. In vitro release and water uptake studies were performed in Dulbecco's phosphate buffered saline at 37 degrees C and 15 rpm. Drug loading and release rates were determined by high performance liquid chromatography. Release studies of Dex and AsAP showed that, after an initial burst release lasting 4 and 9 days, respectively, release rates followed zero order kinetics with high correlation coefficients at least until 35 days. Incorporation of AsAP into the scaffolds increased the release rates of Dex and AsAP, and the scaffold water uptake. When mesenchymal stem cells (MSCs) were cultured in the AsAP and Dex containing scaffolds in vitro, the amount of mineralization was significantly higher than in control scaffolds. In conclusion, AsAP and Dex were incorporated into porous PLGA scaffolds and continuously released over a month and osteogenesis of MSCs was increased by culture in these scaffolds.