Blockade of fatty acid signalling inhibits lipopolysaccharide-induced macrophage recruitment and progression of apical periodontitis.

AIM: To examine the role of palmitic acid in lipopolysaccharide (LPS)-stimulated chemotaxis of macrophages and the potential contribution of saturated fatty acid in signalling during the pathogenesis of apical periodontitis. METHODOLOGY: J774, a mouse macrophage cell line, was used in the experiments. After treatment with LPS, proteolytic maturation of sterol regulatory element-binding protein-1c (SREBP-1c) and expression of fatty acid synthase (FASN) were examined by Western analysis. Levels of palmitic acid were measured by reverse phase-high performance liquid chromatography-mass spectrometry. Knockdown of SREBP-1c and FASN was accomplished by small interfering RNA technology. Secretion of CC-chemokine ligand 2 (CCL2) and cellular chemotaxis were assessed by enzyme-linked immunosorbent assay and transwell migration assay, respectively. Sulfo-N-succinimidyl oleate (SSO) treatment was used to inhibit fatty acid signalling in vitro and also in a rat model of apical periodontitis. All data were first subjected to Levene's test. In vitro data were then analysed using ANOVA followed by Tukey's multiple comparison test. Data from animal experiments were analysed by independent t-tests. The significant level was set at 0.05. RESULTS: LPS stimulated proteolytic maturation of SREBP-1c and FASN expression in macrophages and significantly enhanced palmitic acid synthesis (P < 0.05). Knockdown of SREBP-1c attenuated LPS-enhanced FASN expression. Knockdown of FASN significantly suppressed LPS-enhanced palmitic acid synthesis (P < 0.05). LPS and exogenous palmitic acid significantly enhanced CCL2 secretion and macrophage chemotaxis (all P < 0.05). Inhibition of FASN expression significantly alleviated LPS-augmented CCL2 secretion (P < 0.05). SSO significantly suppressed CCL2 secretion and macrophage chemotaxis augmented by LPS and palmitic acid (all P < 0.05). In a rat model of induced apical periodontitis, SSO treatment significantly attenuated progression of apical periodontitis and macrophage recruitment (all P < 0.05). CONCLUSIONS: LPS/SREBP-1c/FASN/palmitic acid signalling contributed to tissue destruction caused by bacterial infection. Modulation of lipid metabolism and signalling may be helpful for the management of apical periodontitis.

Simvastatin alleviates bone resorption in apical periodontitis possibly by inhibition of mitophagy-related osteoblast apoptosis.

AIM: To assess the connection between mitophagy and hypoxia-induced apoptosis in osteoblasts and whether simvastatin alleviates bone resorption in apical periodontitis through modulation of mitophagy-related apoptosis. METHODOLOGY: Hypoxia-induced generation of reactive oxygen species in mitochondria and changes in mitochondrial membrane potential were evaluated, respectively, by MitoSOX and JC-1 fluorescence dye signalling. Accumulation of mitophagy markers PTEN-induced putative kinase 1 (PINK1) and Parkin in mitochondria was examined by Western blotting and immunofluorescence microscopy. Osteoblast apoptosis was assessed by Western analysis of cleaved-poly (adenosine diphosphate ribose) polymerase (PARP). In a rat model of induced apical periodontitis, the therapeutic effect of simvastatin and its action on osteoblast mitophagy and apoptosis were examined. anova, Fisher's and Student's t-test were used for data analysis. RESULTS: Hypoxia-induced mitochondrial dysfunction and stimulated mitophagy in osteoblasts. Hypoxia also provoked apoptosis in osteoblasts and inhibition of mitophagy decreased hypoxia-augmented apoptotic activity. Simvastatin alleviated hypoxia-induced mitochondrial dysfunction, mitophagy and apoptosis. The protective action of simvastatin against apoptosis was related to its antimitophagy activity. Experiments in the rat model of induced apical periodontitis supported the laboratory findings. Simvastatin treatment mitigated periapical bone loss and reduced the activities of apoptosis and mitophagy in regional osteoblasts. CONCLUSIONS: The results suggest that modulation of osteoblast mitophagy may help diminish bone loss associated with inflammation and has potential as an auxiliary therapy for apical periodontitis.

Changes in Renal Function After Heart Transplantation.

Renal function after heart transplantation (HTx) typically follows a biphasic pattern and an initial decay within 1 to 2 years. Trajectory of renal function after HTx is less reported, especially in Asia. The aims of this cohort study were to describe the changes in HTx recipients' serum creatinine and estimated glomerular filtration rate (eGFR) levels 5 years following HTx in Taiwan. METHODS: We retrospectively reviewed 5 years of 440 consecutive adult patients (≥ 18 years) who underwent first HTx from June 1987 to December 2014 at the National Taiwan University Hospital. RESULTS: Among 422 participants, they received induction therapy consisting of intravenous rabbit antithymocyte globulin. Here, we illustrated the trends over the years by dividing the subjects into 2 groups based on their immunosuppressive regimen of transplantation (1987-2002 and 2003-2014) The pretransplantation median serum creatinine concentration level was 1.2 mg/dL, rose to 1.4 mg/dL at 3 months after surgery, and remained steady over 5 years after HTx. Pretransplant median eGFR was 67 mL/min/1.73 m2.The median serum creatinine concentration level and eGFR at baseline were all significantly difference than pretransplantation (P > .05). This result has showed that an initial steep decline within 3 months after transplant remained stable 5 years after HTx. CONCLUSION: As renal function deteriorates after HTx, we observed a steep decline in serum creatinine level and glomerular filtration rate within the 3 months after HTx, followed by a slow rate of deterioration over the following months. We found a time-related progressive deterioration in renal function during the 5 years after HTx.

Case Series: Heart Transplantation After Fontan Operation-Single-Center Experience.

BACKGROUND: Fontan failure (FF) occurs rarely. In patients with Fontan failure, heart transplantation is believed to be the most effective therapy. We review our experience in heart transplantations after the Fontan operation. METHODS: From July 1987 to December 2014, 4 of 513 patients underwent orthotopic heart transplantation (OHT). Among them, 4 were due to FF. We reviewed these 4 cases via retrospective chart review. Clinical history, laboratory data, surgical technique, perioperative variables, and outcomes of long-term follow-up are presented herein. The primary outcomes were hospital mortality, 1-year-survival rate, and 4-year-survival rate. The secondary outcome is the improvement in patients with protein-losing enteropathy. RESULTS: The hospital mortality rate was 0% in the 4 FF patients receiving OHT. No surgically related hemorrhage or infection was observed. The 1-year-survival rate was 100% (n = 4) and the 4-year-survival rate 50% (n = 2). One patient died of posttransplantation lymphoproliferative disorder. Hypoalbuminemia improved in 1 of 3 patients 4 months after OHT. CONCLUSIONS: Despite technical challenges, heart transplantation can be performed successfully in patients with Fontan operation. However, protein-losing enteropathy might not be resolved quickly after heart transplantation.

Malignancy After Heart Transplantation Under Everolimus Versus Mycophenolate Mofetil Immunosuppression.

BACKGROUND: With advances in immunosuppressive therapy, heart transplantation is currently recommended as the only established surgical treatment for refractory heart failure. However, chronic immunosuppression increases the risk for malignancy. Everolimus (EVR) is a potent mammalian target of rapamycin inhibitor that is used after transplantation and to treat advanced malignancies, as we have done in Taiwan after heart transplantation since 2004. Mycophenolate mofetil (MMF) and EVR are frequently used as cell-cycle inhibitors to optimize post-transplantation outcomes. METHODS: We retrospectively analyzed the characteristics and outcomes of 454 patients who received either MMF (n = 232) or EVR (n = 222) after heart transplantation at the National Taiwan University Hospital from March 1, 1990, to March 1, 2015. Patient characteristics and Kaplan-Meier survival curves were compared between groups. RESULTS: During a median follow-up of 69.2 months, malignancy was diagnosed in 27 patients receiving MMF (n = 23) or EVR (n = 4). There was a significant difference in malignancy risk between groups (9.91% vs 1.80%, P = .001). The most common malignancies were non-Hodgkin lymphoma, skin cancers, and lung squamous cell carcinoma. The 2-year overall survival after malignancy was 50% in the EVR group and 47% in the MMF group (P = .745). CONCLUSIONS: EVR treatment after heart transplant is associated with a lower risk of malignancy than is MMF treatment. The 2-year survival rate after malignancy was similar between EVR and MMF groups.

Low Incidence of Malignancy After Heart Transplantation in Taiwan.

BACKGROUND: Malignancy is the leading cause of death in Taiwan. The risk of malignancy is higher in heart transplant recipients than in the general population. We reviewed the malignancy incidence among the patients who underwent heart transplantation (HT) at the National Taiwan University Hospital (NTUH) during the past 28 years. We found that the incidence of malignancy is low in Taiwan and that the pattern of malignancy is different from that in the Western population. METHODS: From July 1987 to March 2015, 518 patients underwent HT at NTUH. Forty-four patients who died within 1 month after transplantation were excluded from this study. Thus, a total of 476 patients were enrolled in this study. There were 393 male and 83 female patients, with a mean age of 45 years at transplantation. The major indications for HT were dilated cardiomyopathy (52%) and ischemic cardiomyopathy (33%). After HT, all patients received triple immunosuppressive therapy, including a calcineurin inhibitor (cyclosporine or tacrolimus), cell-cycle inhibitor (azathioprine, mycophenolate mofetil, or everolimus), and steroid. After 1995, induction with rabbit anti-human thymocyte globulin was routinely performed. Survival was estimated by means of the Kaplan-Meier method. RESULTS: Twenty-seven patients without pre-transplantation malignancy developed malignancies after HT. The median survival time (MST) of these 27 HT patients was 76.8 months. After malignancy was diagnosed, the overall MST was 20.7 months. The 3- and 5-year overall survival rates were 44% and 27%, respectively. Twenty-one patients (77.8%) died, 10 of them because of cancer. The most common malignancy was non-Hodgkin lymphoma (n = 6), followed by skin cancer (including 2 keratoacanthomas, 2 squamous cell carcinomas, and 1 basal cell carcinoma; n = 5) and lung squamous cell carcinoma (n = 3). The univariate analysis identified cancer stage (P = .044) and comorbidity (P = .002) as factors associated with poor malignancy survival. In the multivariate analysis, comorbidity was an independent prognostic factor for greater risk of death because of post-transplantation malignancy (P = .002). CONCLUSIONS: In Taiwan, the risk of malignancy after HT is low (5.7%), as is the incidence of skin cancer. The most common malignancy was non-Hodgkin lymphoma, followed by skin cancer and lung cancer. Comorbidity was an independent factor for overall survival in cancer patients who previously underwent HT.

Surveillance of Immunosuppression During Pregnancy After Heart Transplantation: Case Report.

BACKGROUND: Transplantation and immunosuppressive drugs are major limitations to the success of pregnancy. In 1988, the first pregnancy after a heart transplant was reported, which has given female recipients the hope to give birth. During pregnancy, physiologic changes with increased blood volume and hemodilution may influence blood drug level. CASE REPORT: We reported our experience in monitoring on immunosuppressive drugs for 2 cases. Both of them underwent heart transplantation in 2006 and were 34 and 37 years old at time of pregnancy. For both cases, we frequently monitored the blood level and increased the dosage of immunosuppressive drugs accordingly. Both cases had uneventful pregnancy and delivery to healthy babies at the National Taiwan University Hospital in Taiwan. Their postpartum courses were uneventful as well. CONCLUSIONS: We advocate adjusting the immunosuppressive dosage according to the blood level before pregnancy.

Current status of heart transplantation in Taiwan.

PURPOSE: We reviewed the national results of heart transplantation in Taiwan. METHODS: From July 1987 to December 2012, 1354 patients underwent heart transplantation in 18 qualified heart centers in Taiwan. The transplantation volume and survival rate were reviewed. RESULTS: The median age of recipients was 49 years at surgery, with 37% in the International Society for Heart and Lung Transplantation (ISHLT)-1A, 30% in ISHLT-1B, and 32% in ISHLT-2. The allograft 1-, 3-, 5-, and 10-year survival rates were 78%, 68%, 61%, and 47%, respectively. Mostly difficult recipients were bridged by extracorporeal membrane oxygenation (ECMO) instead of ventricular assist device (VAD). CONCLUSION: The results of heart transplantation in Taiwan are comparable with ISHLT world results. In Taiwan, we use more ECMO than VAD for mechanical circulatory support to bridge critical recipients to heart transplantation.

Antibody-mediated rejection after orthotopic heart transplantation: a 9-year single-institution experience.

OBJECTIVE: Over the past decade, antibody-mediated rejection (AMR) continues to be recognized as one of the major obstacles in cardiac transplantation, yet its clinical outcome has been reported only in small series studies. This investigation reviews our experience in treating 11 patients with AMR after heart transplantation. METHODS: We retrospectively analyzed a total of 11 patients who underwent cardiac transplantation from 2004 to 2012 at a single medical institute. The diagnosis of AMR was made according to criteria set by the International Society for Heart and Lung Transplantation (ISHLT) 2011 working formulation. RESULTS: The average age among the 11 patients was 50.4 ± 16.9 years. The overall mortality rate was 54.5%. Five patients (45.4%) developed hemodynamic compromise in an average of 5 days after transplantation, presenting with sudden onset of fatal arrhythmia (n = 4; 80%) and immediate heart failure (n = 1; 20%). All 5 patients underwent immediate resuscitation and extracorporeal membrane oxygenation (ECMO) support, and 3 patients died (60%); in contrast, the other 6 patients suffered from progressively worsening cardiac function during long-term follow-up. Three patients (50%) died in this group. CONCLUSIONS: Clinical presentation of AMR varies. Long-term postoperative follow-up in the form of endomyocardial biopsy is recommended with immunohistochemistry C4d staining, with the anticipation of the possibility of future recurrence.

Clinical experience of tacrolimus with everolimus in heart transplantation.

BACKGROUND: Tacrolimus (Tac) in combination with mycophenolate mofetil is widely used after heart transplantation (HT). Everolimus (EVR), a new potent proliferation signal inhibitor can be used with a carcineurin inhibitor to reduce the occurrence of rejection. The purpose of this study was to evaluate the efficacy and safety of Tac combined with EVR in de novo HT. MATERIALS AND METHODS: From January 2009 to April 2011, 33/62 patients who underwent HT were prescribed Tac and EVR as de novo immunosuppression. The main exclusion criteria were poor kidney function (serum creatinine > 2.8 mg/dL), panel-reactive antibodies > 25%, donors > 60 years old, or cold ischemia time > 6 hours. All patients received Tac (C0 blood level 5-10 ng/mL during the first 6 months, then 3-5 ng/mL), EVR (C0 target 3-8 ng/mL), and corticosteroids. After transplantation, routine examinations included echocardiogram and protocol endomyocardial biopsy. RESULTS: There was no operative mortality. The 1- and 3-year actuarial survivals were 95.74% ± 3.49%. One patient who had undergone coronary artery bypass grafting previously and received intra-aortic balloon pumping and extracorporeal membrane oxygenator-assisted cardiopulmonary resuscitation before HT died of Aspergillus septicemia 58 days after HT. No biopsy-proven acute rejection > grade 2R or acute rejection associated with hemodynamic compromise was observed. Hyperlipemia was noted in 16 cases (48.5%), hypertension in 11 (33.3% 5%), and diabetes mellitus in 12 (36.4%). No other severe adverse events were noted. CONCLUSIONS: Concentration-controlled EVR (C0 target 3-8 ng/mL) in combination with Tac achieved good efficacy and safety. The 1- and 3-year actuarial survivals were 95.74% ± 3.49%.

Can cyclosporine blood level be reduced to half after heart transplantation?

BACKGROUND: Cyclosporine (CsA) is widely used after heart transplantation. The purpose of this prospective randomized study was to evaluate the safety and efficacy of reduction of CsA blood level to one-half of the traditional blood concentration under a regimen of everolimus (EVL), CsA, and steroid. MATERIALS AND METHODS: This prospective, 6 month, randomized, open-label study included adult (aged 18 to 65 years) recipients of a primary heart transplant with serum creatinine60 years old, had cold ischemia time>6 hours, or had plasma renin activity>or=25%. All patients received CsA (C2 blood level 1000-1400 ng/mL), EVL (C0 target 3-8 ng/mL), and corticosteroids to day 60, before random entry into one of 2 groups: SE (C2 blood level from days 60-149=800-1200 ng/mL, and days 150-180 C2=600-1000 ng/mL), or RE group with CsA reduced by one-half after 3 months (days 90-149 C2=400-600 ng/mL, and from days 150-180 C2=300-500 ng/mL). RESULTS: The 25 recipients eligible for this study included 13 patients in the SE and 12 in the RE group. There was no operative mortality in either group. No death or graft loss was noted within 6-months in either group. Mean serum creatinine at month 6 tended to be lower in the RE cohort (1.23+/-0.44 mg/dL versus 1.55+/-0.85 mg/dL; P=.093). Biopsy-proven acute rejection>or=grade 3A was observed in only 1 patient (7.7%), who was in the SE group. There were no acute rejection episodes associated with hemodynamic compromise. The incidences of adverse events in each group were similar. CONCLUSIONS: Concentration-controlled EVL (C0 target 3-8 ng/mL) in combination with reduced CsA exposure of one-half the usual concentration achieved good efficacy and safety over 6 months. The renal function at 6 months among the RE group showed a trend toward improvement, suggesting a benefit of halving the target CsA blood level after heart transplantation.

The survival of heart transplant recipients using cyclosporine and everolimus is not inferior to that using cyclosporine and mycophenolate.

INTRODUCTION: Heart transplantation has become the best available therapy for patients with refractory end-stage heart failure. Cyclosporine (CsA) and mycophenolate mofetil (MMF) are the 2 FDA-approved drugs to prevent posttransplant acute rejection episodes. The purpose of this study was to evaluate the result of heart transplantation treated with CsA and everolimus (EVL), compared with that of patients treated with CsA and MMF. MATERIALS AND METHODS: From 2000 to 2009 heart transplantation was performed in 239 patients among whom we enrolled 93 patients with a serum creatinine values

Disseminated peritoneal leiomyomatosis responds to systemic chemotherapy.

Leiomyomatosis peritonealis disseminata (LPD) is a rare disease entity characterized by multiple peritoneal tumors composed of benign but proliferative smooth muscle cells. Surgery is the mainstay treatment for LPD. We present a 50-year-old woman who had previously undergone several surgical resections including bilateral oophorectomy for recurrent LPD. Because of progressive tumors in the peritoneum and metastatic tumors in the liver and lungs, systemic chemotherapy with doxorubicin and dacarbazine was prescribed. Objective tumor response was achieved and sustained for 1 year. This case presentation suggests that systemic chemotherapy may be considered as a treatment option for LPD patients developing unresectable or metastatic disease.

Differential expression of glucocorticoid receptor in carcinomas of the human digestive system.

AIMS: To investigate the in situ expression profile of glucocorticoid receptor (GR) in normal and carcinomatous tissues of the human digestive system. METHODS AND RESULTS: Specimens from 306 carcinomas of the human digestive tract were assayed for the expression of GR by immunohistochemistry. GR expression was strong in oesophageal squamous epithelia, pancreatic islet cells and hepatocytes, but generally weak or negative in non-squamous epithelia. Consistently, GR expression was found in a high percentage of oesophageal squamous cell carcinomas (SCC) (98.1%) and hepatocellular carcinomas (HCC) (92.9%), but rarely in gastric adenocarcinomas (7.4%) and not at all in colorectal adenocarcinomas (0%). Dexamethasone (DEX) was found to confer chemoresistance in oesophageal SCC and HCC cells, suggesting that GR expression may be biologically important in some GR-expressing carcinomas. CONCLUSIONS: Distribution of GR expression is markedly diverse among tissues of the human digestive system. The general lack of GR in adenocarcinomas contrasts with the high percentage of SCCs and HCCs expressing GR, and, along with the generation of chemoresistance by DEX, warrants prospective study of the effects of steroids on these cancers.

Derivation, characterization and differentiation of human embryonic stem cells: comparing serum-containing versus serum-free media and evidence of germ cell differentiation.

BACKGROUND: This study was designed to establish human embryonic stem cell (hESC) lines, to identify the differences when maintained in serum-containing versus serum-free medium and to test their potential of in vitro differentiation. METHODS: Procedures including immunosurgery were performed on 11 donated human blastocysts to establish hESC lines. The cell lines were characterized and maintained using either serum-free or serum-containing media to compare their morphology, Oct-4 expression, apoptosis and growth speed. Differentiation of these lines was evaluated by the morphology and the expression of genes belonging to the three embryonic germ layers and the germ cell lineage. RESULTS: Three hESC lines were established, and they grew at similar speed in both media (serum-containing or serum-free), but hESC cultured in serum-containing medium yielded significantly higher percentages of morphologically good colonies and cells expressing Oct-4. These cell lines differentiated spontaneously in vitro into cells expressing markers belonging to all three embryonic germ layers and germ cell markers, including c-Kit, STELLA, VASA and growth differentiation factor 9 (GDF9), in directly adherent culture. CONCLUSIONS: Three hESC lines with Taiwanese ancestry have been established, and they retain the in vitro differentiation potential with or without embryoid body (EB) formation. The data support that hESC may be capable of differentiation into germ cells although further confirmation is needed. It is also suggested that strategies such as stepwise adaptation will be needed before implementing a serum-free culture condition for hESC lines that have previously been derived in a medium containing serum.

Effect of plasmapheresis for acute humoral rejection after heart transplantation.

OBJECTIVE: The objective of this study was to report our experience of treating acute humoral rejection with plasmapheresis in heart transplant (HT) recipients. PATIENTS AND METHODS: From May 1996 to December 2005, 238 HTs were performed using therapy with cyclosporine or tacrolimus, azathioprine or mycophenolate mofetil, and prednisolone as well as induction treatment with rabbit anti-human thymocyte globulin. Endomyocardial biopsy for rejection surveillance was performed weekly for the first month, monthly for 3 months, yearly after the first year, and whenever rejection was suspected. Immunofluorescence studies with IgG, IgM, C3, C4, C1q, and HLA-DR were performed routinely on the first month biopsy. After a 2-year trial, immunofluorescence studies were not performed routinely, because no significant findings were observed; thus they were performed only when clinical deterioration, unstable hemodynamic status, or suspicion of rejection occurred on routine echocardiographic examinations. Plasmapheresis with fresh frozen plasma exchanging twice the blood volume of the patients was performed for 5 days. Rescue immunosuppression with methylprednisolone (1 g/d) was delivered for 3 days and the immunosuppressants changed, but no intravenous immunoglobulin was prescribed. RESULTS: Twelve patients suffered biopsy-proven acute humoral rejection at 3 days to 32 months after HT (mean, 9.4 months). Immunofluorescence studies showed positive HLA-DR in 7 patients; IgG in 4 patients; IgM in 1 patient; C3 in 4 patients; C4 in 1 patient; and C1q in 1 patient. One patient who was 3 months after HT showed only C1q positive but was treated with extracorporeal membrane oxygenation and intra-aortic balloon pumping support and died 1-month after plasmapheresis. Another patient who deteriorated on the 3rd postoperative day and died 3 days after plasmapheresis was considered to have vascular rejection by interstitial edema, vacuolated endothelial cells and no pathognomonic clinical features, although there was no positive immunofluorescence result. All other subjects were discharged from the hospital, although 3 required mechanical support during plasmapheresis. Hypotension with hypocalcemia was frequently noted during plasmapheresis. The 1-year survival rate was 75% +/- 11%, and 5-year survival rate, 51% +/- 15%. CONCLUSION: Plasmapheresis with concurrent rescue immunosuppression was an effective treatment for acute humoral rejection in HT even with unstable hemodynamics.

Placenta growth factor expression is correlated with survival of patients with colorectal cancer.

BACKGROUND: Overexpression of vascular endothelial growth factor (VEGF) correlates with vascularity, metastasis, and proliferation in colorectal cancer but the role of its homologue, placenta growth factor (PlGF), is unknown. The aim of this study was to evaluate expression and clinical implications of PlGF in colorectal cancer. METHODS: We investigated 74 tumour/non-tumour pairs of colorectal cryosections. Clinical staging was based on the UICC-TNM classification. Expression levels of mRNA for PlGF and VEGF were analysed with quantitative real time reverse transcription-polymerase chain reaction. Proteins were analysed by immunohistochemical staining and enzyme linked immunoabsorbant assay. Analysis of the differences in PlGF and VEGF levels between tumour and non-tumour tissues in the same patient were performed by paired t test; differences between localised and advanced disease patients by the Mann-Whitney, chi(2), and Fisher's exact tests and survival curves by the Kaplan-Meier method. RESULTS: Expression levels for both growth factors were significantly higher in tumour than in non-tumour tissues (p