Tunable multi-responsive nano-gated mesoporous silica nanoparticles as drug carriers.

Tunable multi-responsive mesoporous silica nanoparticles were prepared by post-condensation/surface modification of MCM-41 nanoparticles. Surface grafting of a poly(N,N-dimethylaminoethyl methacrylate)-based polymer containing disulfide bonds was achieved by a click reaction. Chemical modification, morphological characteristics, and textural properties of the nanoparticles were studied using multiple characterization techniques such as Fourier transform infrared spectroscopy, thermogravimetric analysis, scanning electron microscopy, transmission electron microscopy, small-angle X-ray scattering, and nitrogen adsorption/desorption behavior. The nanoparticles retained the meso-structural integrity of MCM41 and particle size < 100 nm after grafting with the polymer. The pH and redox-responsive behavior of the nanoparticles were also studied. The nanoparticles possess excellent drug-loading capacity owing to their large surface area and 'closed gate' mechanism of the grafted polymer chains. The release profile of doxorubicin at two different pH (7.4 and 5.5) and in the presence of dithiothreitol showed a dual response behavior. The nano drug carrier device exhibited efficient intracellular uptake in cancer cells with suitable cytotoxicity and pharmacokinetic behavior, and may therefore be considered a good candidate for cancer therapy.

Potentiation of TRAIL­induced cell death by nonsteroidal anti­inflammatory drug in human hepatocellular carcinoma cells through the ER stress­dependent autophagy pathway.

Hepatocellular carcinoma (HCC) is the most commonly diagnosed primary liver malignancy. The limited success with relapse of the disease in HCC therapy is frequently associated with the acquired resistance to anticancer drugs. To develop a strategy and design for overcoming the resistance of HCC cells to TNF­related apoptosis inducing ligand (TRAIL)­induced cell death, we evaluated the efficacy of a non­steroidal anti­inflammatory drug (NSAID) in combination with TRAIL against TRAIL­resistant HCC cells expressing a high level of CD44. We revealed by MTT and western blotting, respectively, that celecoxib (CCB), an NSAID, and 2,5­dimethyl celecoxib (DMC), a non­cyclooxygenase (COX)­2 inhibitor analog of CCB, were able to sensitize TRAIL­resistant HCC cells to TRAIL, implicating a COX­independent mechanism. CCB dose­dependently enhanced LC3­II and reduced p62 levels through AMPK activation and inhibition of the Akt/mTOR pathway and upregulated expression of ATF4/CHOP, leading to activation of endoplasmic reticulum (ER) stress­dependent autophagy. The TRAIL sensitization capacity of CCB in TRAIL­resistant HCC cells was abrogated by an ER stress inhibitor. In addition, we also revealed by flow cytometry and western blotting, respectively, that accelerated downregulation of TRAIL­mediated c­FLIP expression, DR5 activation and CD44 degradation/downregulation by NSAID resulted in activation of caspases and poly(ADP­ribose) polymerase (PARP), leading to the sensitization of TRAIL­resistant HCC cells to TRAIL and thereby reversal of TRAIL resistance. From these results, we propose that NSAID in combination with TRAIL may improve the antitumor activity of TRAIL in TRAIL­resistant HCC, and this approach may serve as a novel strategy that maximizes the therapeutic efficacy of TRAIL for clinical application.

Nonsteroidal Anti-inflammatory Drugs Sensitize CD44-Overexpressing Cancer Cells to Hsp90 Inhibitor Through Autophagy Activation.

Recently, novel therapeutic strategies have been designed with the aim of killing cancer stem-like cells (CSCs), and considerable interest has been generated in the development of specific therapies that target stemness-related marker of CSCs. In this study, nonsteroidal anti-inflammatory drugs (NSAIDs) significantly potentiated Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG)-mediated cytotoxicity through apoptotic and autophagic cell death induction, but COX-2-inhibitory function was not required for NSAID-induced autophagy in CD44-overexpressing human chronic myeloid leukemia K562 (CD44highK562) cells. Importantly, we found that treatment with NSAIDs resulted in a dose-dependent increase in LC3-II level and decrease in p62 level and simultaneous reduction in multiple stemness-related markers including CD44, Oct4, c-Myc, and mutant p53 (mutp53) in CD44highK562 cells, suggesting that NSAIDs could induce autophagy, which might mediate degradation of stemness-related marker proteins. Activation of AMPK and inhibition of Akt/mTOR/p70S6K/4EBP1 participated in NSAID-induced autophagy in CD44highK562 cells. In addition, treatment of CD44highK562 cells with NSAIDs inhibited expression of HSF1/Hsps, which resulted in suppression of 17-AAG-induced activation of Hsp70, leading to reversal of 17-AAG resistance and sensitization of CD44highK562 cells to 17-AAG by NSAIDs. In conclusion, combining NSAIDs with Hsp90 inhibitor may offer one of the most promising strategies for eradication of CD44-overexpressing CSCs.

Sensitization of multidrug-resistant cancer cells to Hsp90 inhibitors by NSAIDs-induced apoptotic and autophagic cell death.

NSAIDs (non-steroidal anti-inflammatory drugs) have potential use as anticancer agents, either alone or in combination with other cancer therapies. We found that NSAIDs including celecoxib (CCB) and ibuprofen (IBU) significantly potentiated the cytotoxicity of Hsp90 inhibitors in human multidrug-resistant (MDR) cells expressing high levels of mutant p53 (mutp53) protein and P-glycoprotein (P-gp), and reversed Hsp90 inhibitor resistance caused by activation of heat shock factor 1 (HSF1) and by up-regulation of heat shock proteins (Hsps) and P-gp. Inhibition of Akt/mTOR and STAT3 pathways by CCB induced autophagy, which promoted the degradation of mutp53, one of Hsp90 client proteins, and subsequently down-regulated HSF1/Hsps and P-gp. Inhibition of autophagy prevented mutp53 degradation and CCB-induced apoptosis, and inhibition of caspase-3-mediated apoptotic pathway by Z-DEVD-FMK did not completely block CCB-induced cell death in MDR cells, suggesting that autophagic and apoptotic cell death may contribute to CCB-induced cytotoxicity in MDR cells. Furthermore, CCB and IBU suppressed Hsp90 inhibitor-induced HSF1/Hsp70/P-gp activity and mutp53 expression in MDR cells. Our results suggest that NSAIDs can be used as potential Hsp90 inhibitor chemosensitizers and reverse resistance of MDR cells to Hsp90 inhibitors via induction of apoptosis and autophagy. These results might enable the use of lower, less toxic doses of Hsp90 inhibitors and facilitate the design of practically applicable, novel combination therapy for the treatment of MDR cancer.

Decreased Muc5AC expression is associated with poor prognosis in gastric cancer.

Mucins reportedly play numerous key roles in carcinogenesis, including in tumor invasion, regulation of differentiation and tumor cell proliferation. We investigated the effect of Muc5AC, a secreted mucin, on the invasiveness/migratory capability of gastric cancer cells and the prognostic significance of Muc5AC in gastric cancer patients. The clinicopathological and prognostic significance of Muc5AC expression was validated using immunohistochemical analysis in 412 gastric cancer patients. Differential gene expression was investigated using complementary DNA microarray analysis of 48 fresh tumor tissue samples. Silencing of Muc5AC by using a small hairpin RNA-containing lentivirus increased the invasion and migration of SNU216 and AGS cells as well as Akt phosphorylation and the expression of vascular endothelial growth factor and matrix metalloproteinase-7, which were blocked by inhibitors of the phosphatidylinositol 3-kinase/Akt pathway. Loss of Muc5AC expression was significantly associated with tumor progression (advanced T stage; p = 0.004), lymph node metastases (p = 0.001), lymphovascular invasion (p < 0.0001), and increased tumor size (p = 0.027). Lower MUC5AC expression was identified as an independent poor prognostic factor in diffuse-type gastric cancer by using the Cox regression proportional hazard model (hazard ratio, 2.39; p = 0.043). Complementary DNA microarray analysis revealed 86 differentially expressed genes, including genes related to metastasis and invasion, in gastric cancer tissues with high (≥25%) and low (<25%) Muc5AC expression levels. Low Muc5AC expression increased the invasion and migration of gastric cancer cells and could be a useful biomarker of poor prognosis in gastric cancer.

S100A8 and S100A9 promotes invasion and migration through p38 mitogen-activated protein kinase-dependent NF-κB activation in gastric cancer cells.

S100A8 and S100A9 (S100A8/A9) are low-molecular weight members of the S100 family of calcium-binding proteins. Recent studies have reported S100A8/A9 promote tumorigenesis. We have previously reported that S100A8/A9 is mostly expressed in stromal cells and inflammatory cells between gastric tumor cells. However, the role of environmental S100A8/A9 in gastric cancer has not been defined. We observed in the present study the effect of S100A8/A9 on migration and invasion of gastric cancer cells. S100A8/ A9 treatment increased migration and invasionat lower concentrations that did not affect cell proliferation and cell viability. S100A8/A9 caused activation of p38 mitogenactivated protein kinase (MAPK) and nuclear factor-κB (NF-κB). The phosphorylation of p38 MAPK was not affected by the NF-κB inhibitor Bay whereas activation of NF-κB was blocked by p38 MAPK inhibitor SB203580, indicating that S100A8/A9-induced NF-κB activation is mediated by phosphorylation of p38 MAPK. S100A8/A9-induced cell migration and invasion was inhibited by SB203580 and Bay, suggesting that activation of p38 MAPK and NF-κB is involved in the S100A8/A9 induced cell migration and invasion. S100A8/A9 caused an increase in matrix metalloproteinase 2 (MMP2) and MMP12 expression, which were inhibited by SB203580 and Bay. S100A8/A9-induced cell migration and invasion was inhibited by MMP2 siRNA and MMP12 siRNA, indicating that MMP2 and MMP12 is related to the S100A8/A9 induced cell migration and invasion. Taken together, these results suggest that S100A8/A9 promotes cell migration and invasion through p38 MAPKdependent NF-κB activation leading to an increase of MMP2 and MMP12 in gastric cancer.

Overexpression of Snail is associated with lymph node metastasis and poor prognosis in patients with gastric cancer.

BACKGROUND: Epithelial-mesenchymal transition (EMT) plays a significant role in tumor progression and invasion. Snail is a known regulator of EMT in various malignant tumors. This study investigated the role of Snail in gastric cancer. METHODS: We examined the effects of silenced or overexpressed Snail using lenti-viral constructs in gastric cancer cells. Immunohistochemical analysis of tissue microarrays from 314 patients with gastric adenocarcinoma (GC) was used to determine Snail's clinicopathological and prognostic significance. Differential gene expression in 45 GC specimens with Snail overexpression was investigated using cDNA microarray analysis. RESULTS: Silencing of Snail by shRNA decreased invasion and migration in GC cell lines. Conversely, Snail overexpression increased invasion and migration of gastric cancer cells, in line with increased VEGF and MMP11. Snail overexpression (≥75% positive nuclear staining) was also significantly associated with tumor progression (P < 0.001), lymph node metastases (P = 0.002), lymphovascular invasion (P = 0.002), and perineural invasion (P = 0.002) in the 314 GC patients, and with shorter survival (P = 0.023). cDNA microarray analysis revealed 213 differentially expressed genes in GC tissues with Snail overexpression, including genes related to metastasis and invasion. CONCLUSION: Snail significantly affects invasiveness/migratory ability of GCs, and may also be used as a predictive biomarker for prognosis or aggressiveness of GCs.

Identification of S100A8 and S100A9 as negative regulators for lymph node metastasis of gastric adenocarcinoma.

With increasing therapeutic use of minimally invasive therapy for treatment of early gastric cancer, prediction of lymph node metastasis is important. In search of tissue biomarkers for prediction of lymph node metastasis of gastric adenocarcinoma, we analyzed gastric adenocarcinoma tissue using proteomic methods. We have done 2D-PAGE and MALDI-TOF MS analysis in matched normal and gastric cancer tissues. We also evaluated the clinicopathological significance of expression of S100A8 and S100A9 in gastric adenocarcinoma using a tissue microarray of 218 gastric adenocarcinoma specimens. Cell invasion and migration assay were performed to confirm functional role of S100A8 and S100A9 using small hairpin RNA lentivirus. We identified 8 up-regulated and 5 down-regulated proteins in gastric cancer tissues compared to matched normal mucosa. Of these, expression of S100A8 and S100A9 occurred mainly in stromal cells and inflammatory cells between tumor cells. Correlation was observed between small lesion size, decreased depth of invasion, a tendency to absence of lymphovascular tumor emboli, a decrease in perineural invasion and lymph node metastasis, and expression of stromal S100A8. In addition, increased expression of stromal S100A9 in gastric adenocarcinoma was associated with small lesion size and a decrease in lymph node metastasis. Functional analysis confirmed that down-regulation of S100A8 and S100A9 by small hairpin RNA lentivirus induced an increase of migration and invasion in gastric cancer cell lines. Taken together, these findings suggest that S100A8 and S100A9 are negative regulators of lymph node metastasis of gastric adenocarcinoma and can be used as biomarkers for prediction of lymph node metastasis in gastric adenocarcinoma.

Primary Anti-retroviral Resistance in Treatment-naive HIV-infected Patients: A Korean HIV/AIDS Cohort Study / 감염과화학요법

Primary anti-retroviral resistance is considered one of the major problems of HIV treatment. Contrary to reports from western countries, prior Korean studies have reported a relatively low primary resistance rate (less than 5%). Based on Korean HIV/AIDS cohort data, we estimated the primary resistance rate among treatment-naive HIV-infected patients. According to the results, the primary resistance rate was higher (8.8%) than reported previously in Korean studies. However, the major PI mutation was not found.

Primary Anti-retroviral Resistance in Treatment-naive HIV-infected Patients: A Korean HIV/AIDS Cohort Study / 감염과화학요법

Primary anti-retroviral resistance is considered one of the major problems of HIV treatment. Contrary to reports from western countries, prior Korean studies have reported a relatively low primary resistance rate (less than 5%). Based on Korean HIV/AIDS cohort data, we estimated the primary resistance rate among treatment-naive HIV-infected patients. According to the results, the primary resistance rate was higher (8.8%) than reported previously in Korean studies. However, the major PI mutation was not found.

Cancer-derived lysophosphatidic acid stimulates differentiation of human mesenchymal stem cells to myofibroblast-like cells.

Lysophosphatidic acid (LPA) is enriched in ascites of ovarian cancer patients and is involved in growth and invasion of ovarian cancer cells. Accumulating evidence suggests cancer-associated myofibroblasts play a pivotal role in tumorigenesis through secreting stromal cell-derived factor-1 (SDF-1). In the present study, we demonstrate that LPA induces expression of alpha-smooth muscle actin (alpha-SMA), a marker for myofibroblasts, in human adipose tissue-derived mesenchymal stem cells (hADSCs). The LPA-induced expression of alpha-SMA was completely abrogated by pretreatment of the cells with Ki16425, an antagonist of LPA receptors, or by silencing LPA(1) or LPA(2) isoform expression with small interference RNA (siRNA). LPA elicited phosphorylation of Smad2/3, and siRNA-mediated depletion of endogenous Smad2/3 or adenoviral expression of Smad7, an inhibitory Smad, abrogated the LPA induced expression of alpha-SMA and phosphorylation of Smad2/3. LPA-induced secretion of transforming growth factor (TGF)-beta1 in hADSCs, and pretreatment of the cells with SB431542, a TGF-beta type I receptor kinase inhibitor, or anti-TGF-beta1 neutralizing antibody inhibited the LPA-induced expression of alpha-SMA and phosphorylation of Smad2. Furthermore, ascites from ovarian cancer patients or conditioned medium from ovarian cancer cells induced expression of alpha-SMA and phosphorylation of Smad2, and pretreatment of the cells with Ki16425 or SB431542 abrogated the expression of alpha-SMA and phosphorylation of Smad2. In addition, LPA increased the expression of SDF-1 in hADSCs, and pretreatment of the cells with Ki16425 or SB431562 attenuated the LPA-stimulated expression of SDF-1. These results suggest that cancer-derived LPA stimulates differentiation of hADSCs to myofibroblast-like cells and increases SDF-1 expression through activating autocrine TGF-beta1-Smad signaling pathway.

Sphingosylphosphorylcholine stimulates expression of fibronectin through TGF-beta1-Smad-dependent mechanism in human mesenchymal stem cells.

Sphingosylphosphorylcholine (SPC) has been reported to stimulate the expression of fibronectin (FN), which plays a key role in cell recruitment and adhesion during wound healing. In a previous study, we reported that SPC induces differentiation of human adipose tissue-derived mesenchymal stem cells (hATSCs) to smooth muscle-like cell types through ERK-dependent autocrine secretion of TGF-beta1 and delayed activation of the TGF-beta1-Smad pathway. In the present study, we demonstrated that SPC dose- and time-dependently increased the expression of FN in hATSCs. Pretreatment of the cells with U0126, an MEK inhibitor, markedly attenuated the SPC-induced expression of FN and delayed phosphorylation of Smad2, suggesting that ERK is involved in the SPC induction of FN expression through activation of Smad2. In addition, the SPC-induced expression of FN and delayed activation of Smad2 were abrogated by SB-431542, a TGF-beta type I receptor kinase inhibitor, or anti-TGF-beta1 neutralizing antibody. Furthermore, the SPC-induced expression of FN was abrogated by adenoviral expression of Smad7, an inhibitory Smad, or short interference RNA (siRNA)-mediated depletion of endogenous Smad2 expression, suggesting that SPC induces the expression of FN through ERK-dependent activation of the TGF-beta1-Smad2 crosstalk pathway. Adhesion of U937 monocytic cells to hATSCs was enhanced by pretreatment of hATSCs with SPC or TGF-beta1 for 4 days, and the peptide GRGDSP (an antagonist of fibronectin receptors) blocked the adhesion of U937 cells to the hATSCs. These results led us to suggest that SPC-induced FN expression plays a pivotal role in the wound healing by stimulating adhesion and recruitment of leukocytes.

The effect of nanofiller on the opacity of experimental composites.

The purpose of this study was to evaluate the effect of the nanofiller in experimental composites on opacity (contrast ratio). Thirteen experimental composites were prepared with three different sizes of fillers: barium glass minifiller (1 microm; 69-76 wt %), silica microfiller (0.04 microm; 0-6 wt %), and silica nanofiller (7 nm; 0-7 wt %). After disk-type specimens were irradiated with a halogen light curing unit at 500 mW/cm(2) for 30 s, the specimens were aged for 6 h at room conditions and were stored in deionized water for 1, 7, 14, 21, 28, 56, and 84 days. The contrast ratios of the specimens were measured as a function of aging period using a spectrophotometer. The distribution morphology of the filler particles in the resin matrix was also examined using energy-filtering transmission electron microscopy. The experimental composites that contained more than 3% nanofiller had significantly lower contrast ratios (p < 0.05). The composites that contained 6 wt % nanofiller had contrast ratios 34-65% lower than the composite that did not contain nanofiller. The values of the contrast ratio from the composites that excluded microfiller were lower than the values from the composites that included microfiller. From the comparison with the 3 different sizes of filler, the contrast ratio of the composite that contained 70 wt % minifiller and 6 wt % microfiller was the highest, the contrast ratio of the composite that contained only 76 wt % minifiller was the median value, and the contrast ratio of the composite that contained 70 wt % minifiller and 6 wt % nanofiller was the lowest. When the microfiller content was decreased from 6 wt % to 0 wt %, the contrast ratio decreased 6-9%. Energy-filtering transmission electron microscopy images indicated that the contrast ratio of experimental composites is related to the distribution morphology of the filler particles in the resin matrix.

Sphingosylphosphorylcholine induces differentiation of human mesenchymal stem cells into smooth-muscle-like cells through a TGF-beta-dependent mechanism.

Mesenchymal stem cells (MSCs) can differentiate into diverse cell types including adipogenic, osteogenic, chondrogenic and myogenic lineages. In the present study, we demonstrated for the first time that sphingosylphosphorylcholine (SPC) induces differentiation of human adipose-tissue-derived mesenchymal stem cells (hATSCs) to smooth-muscle-like cell types. SPC increased the expression levels of several smooth-muscle-specific genes, such as those for alpha-smooth-muscle actin (alpha-SMA), h1-calponin and SM22alpha, as effectively as transforming growth factor beta (TGF-beta1) and TGF-beta3. SPC elicited delayed phosphorylation of Smad2 after 24 hours exposure, in contrast to rapid phosphorylation of Smad2 induced by TGF-beta treatment for 10 minutes. Pretreatment of the cells with pertussis toxin or U0126, an MEK inhibitor, markedly attenuated the SPC-induced expression of beta-SMA and delayed phosphorylation of Smad2, suggesting that the Gi/o-ERK pathway is involved in the increased expression of alpha-SMA through induction of delayed Smad2 activation. In addition, SPC increased secretion of TGF-beta1 through an ERK-dependent pathway, and the SPC-induced expression of alpha-SMA and delayed phosphorylation of Smad2 were blocked by SB-431542, a TGF-beta type I receptor kinase inhibitor, or anti-TGF-beta1 neutralizing antibody. Silencing of Smad2 expression with small interfering RNA (siRNA) abrogated the SPC-induced expression of alpha-SMA. These results suggest that SPC-stimulated secretion of TGF-beta1 plays a crucial role in SPC-induced smooth muscle cell (SMC) differentiation through a Smad2-dependent pathway. Both SPC and TGF-beta increased the expression levels of serum-response factor (SRF) and myocardin, transcription factors involved in smooth muscle differentiation. siRNA-mediated depletion of SRF or myocardin abolished the alpha-SMA expression induced by SPC or TGF-beta. These results suggest that SPC induces differentiation of hATSCs to smooth-muscle-like cell types through G(i/o)-ERK-dependent autocrine secretion of TGF-beta, which activates a Smad2-SRF/myocardin-dependent pathway.

Sphingosylphosphorylcholine induces proliferation of human adipose tissue-derived mesenchymal stem cells via activation of JNK.

Sphingosylphosphorylcholine (SPC) has been implicated in a variety of cellular responses, including proliferation and differentiation. In this study, we demonstrate that d-erythro-SPC, but not l-threo-SPC, stereoselectively stimulated the proliferation of human adipose tissue-derived mesenchymal stem cells (hADSCs), with a maximal increase at 5 microM, and increased the intracellular concentration of Ca(2+) ([Ca(2+)](i)) in hADSCs, which do not express known SPC receptors (i.e., OGR1, GPR4, G2A, and GPR12). The SPC-induced proliferation and increase in [Ca(2+)](i) were sensitive to pertussis toxin (PTX) and the phospholipase C (PLC) inhibitor U73122, suggesting that PTX-sensitive G proteins, Gi or Go, and PLC are involved in SPC-induced proliferation. In addition, SPC treatment induced the phosphorylation of c-Jun and extracellular signal-regulated kinase, and SPC-induced proliferation was completely prevented by pretreatment with the c-Jun N-terminal kinase (JNK)-specific inhibitor SP600125 but not with the MEK-specific inhibitor U0126. Furthermore, the SPC-induced proliferation and JNK activation were completely attenuated by overexpression of a dominant negative mutant of JNK2, and the SPC-induced activation of JNK was inhibited by pretreatment with PTX or U73122. Treatment of hADSCs with lysophosphatidic acid (LPA) receptor antagonist, Ki16425, had no impact on the SPC-induced increase in [Ca(2+)](i). However, SPC-induced proliferation was partially, but significantly, attenuated by pretreatment of the cells with Ki16425.These results indicate that SPC stimulates the proliferation of hADSCs through the Gi/Go-PLC-JNK pathway and that LPA receptors may be responsible in part for the SPC-induced proliferation.

Acute Respiratory Distress Syndrome Induced by Adenovirus in an Otherwise Healthy Woman

Acute respiratory distress syndrome (ARDS) caused by adenovirus is a rare event in healthy adults, especially in non- military settings. Although treatment with intravenous ribavirin has been reported, supportive care, including mechanical ventilation, is known to be the main stay in the treatment of ARDS caused by adenovirus, with high-dose steroid treatment having rarely been reported. We report our experience with a 41-year-old, otherwise healthy, woman with ARDS, treated with high-dose steroid and mechanical ventilatory support.

The First Successful Heart-Lung Transplantation in Korea / 대한흉부외과학회지

The first heart-lung transplantation in Korea was successfully performed. The recipient was a 11 year old girl with pulmonary atresia with intact ventricular septum. She had been catheterized at the ages of 4 months, 3 years, 7 years and 10 years, which revealed that neither Fontan nor biventricular repair was feasible. The donor was a traffic accident victim, a 9 year-old boy with the same blood type. The donor was pronounced dead according to the guidelines of the Korean Medical Association's Brain Death Committee. The operation was performed on April 20, 1997. The native heart-lung block was explanted segmentally and donor one was placed above the phrenic nerve using the Arizona technique. After the tracheal anastomosis with single continuous 4-0 prolene, both vena cavae were anastomosed, followed by aortic anastomosis. The graft ischemic time was 145 minutes. The postoperative course was complicated by fever and tracheal stenosis at the anastomosis site. The fever was controlled by anti-tuberculous medications and the tracheal stenosis was relieved by stent (Palmaz 8 mm, 30 mm in length) placement on POD #71. The patient is doing well and is very active in her 7th postoperative month.