Optimising utilisation of kidneys from very young deceased donors: the technique of en bloc kidney transplantation.

Kidneys of paediatric deceased donors were previously considered suboptimal for older recipients. An 18-month-old deceased donor was made available via Singapore's Medical (Therapy, Education and Research) Act. To the best of our knowledge, she is the youngest local donor. We herein report a case of successful kidney transplantation, using the en bloc technique, to a 15-year-old girl with renal failure secondary to bilateral cystic dysplastic kidney.

Antioxidants and NOS inhibitors selectively targets manganese-induced cell volume via Na-K-Cl cotransporter-1 in astrocytes.

Manganese has shown to be involved in astrocyte swelling. Several factors such as transporters, exchangers and ion channels are attributed to astrocyte swelling as a result in the deregulation of cell volume. Products of oxidation and nitration have been implied to be involved in the pathophysiology of swelling; however, the direct link and mechanism of manganese induced astrocyte swelling has not been fully elucidated. In the current study, we used rat primary astrocyte cultures to investigate the activation of Na-K-Cl cotransporter-1 (NKCC1) a downstream mechanism for free radical induced astrocyte swelling as a result of manganese toxicity. Our results showed manganese, oxidants and NO donors as potent inducer of oxidation and nitration of NKCC1. Our results further confirmed that manganese (50 µM) increased the total protein, phosphorylation and activity of NKCC1 as well as cell volume (p < 0.05 vs. control). NKCC1 inhibitor (bumetanide), NKCC1-siRNA, antioxidants; DMTU, MnTBAP, tempol, catalase and Vit-E, NOS inhibitor; L-NAME, peroxinitrite scavenger; uric acid all significantly reversed the effects of NKCC1 activation (p < 0.05). From the current investigation we infer that manganese or oxidants and NO induced activation, oxidation/nitration of NKCC1 play an important role in the astrocyte swelling.

Cholangitis in children with biliary atresia: health-care resource utilisation.

AIM: Cholangitis is a well-known complication that contributes to morbidity, mortality, as well as health-care utilisation in children with biliary atresia who have undergone the Kasai portoenterostomy. The aim of the study was to determine the common causative organisms for cholangitis and characterise its burden, health-care resource and service utilisation and cost. METHODS: This was a retrospective chart review of children who underwent Kasai portoenterostomy in our institution from 1988 to 2011. The causative organisms were identified based on culture reports. The burden of the disease was estimated based on the number of patients experiencing one or more episodes of cholangitis. Health-care resource and service utilisation were based on different categories, and cost was computed based on the charges at the institution. RESULTS: Twenty-seven (64.3%) out of 42 children included in the analysis experienced at least one episode of cholangitis. There were a total of 97 episodes of cholangitis, with an average of 3.6 (1-15) episodes per patient. The average length of stay per episode of cholangitis was 14.8 (2-64) days. Common organisms isolated during blood cultures were Klebsiella pneumoniae, Enterococcus, Escherichia coli and Pseudomonas aeruginosa. The estimated cost per in-patient admission of 15 days (rounded off) for a single episode of cholangitis was $SG 8986.61 ($US 7369.02). CONCLUSION: The knowledge about the incidence and cost of cholangitis will allow physicians to counsel parents of children newly diagnosed with biliary atresia and to better prepare them both emotionally and financially for what to expect.

Type 1 Bland Sutton colonic atresia complicated by fetalis hydrops in a premature neonate.

Colonic atresia (CA) is an unusual cause of neonatal intestinal obstruction where a section of the colon has failed to form, leading to blockage or absence. A premature baby was delivered at 32 weeks of gestation via caesarian section following fetal distress. She was grossly oedematous and diagnosed with severe fetalis hydrops secondary to anaemia. She was resuscitated and stabilized. On the sixth day of life, the neonate's abdomen became severely distended with billous vomiting and failure to pass meconium. We suspected intestinal obstruction and performed an omnipaque enema which revealed dilated small bowel loops and a bowel atresia. Exploratory laparotomy confirmed a Type 1 Bland Sutton CA with mucosal web. An end colostomy was successfully performed and uneventful. In our case report, we describe a rare occurrence of postnatally diagnosed CA, complicated by fetalis hydrops and anaemia.

α-Synuclein overexpression enhances manganese-induced neurotoxicity through the NF-κB-mediated pathway.

Exposure to manganese (Mn) occurs in both civilian and military operations. Mn exposure results in a movement disorder termed manganism, which resembles Parkinson's disease (PD). However, the pathogenic mechanisms underlying this disorder are not fully understood. α-Synuclein, a presynaptic protein is implicated in some neurodegenerative disorders, including PD and Mn-induced apoptosis, and its overexpression contributes to the loss of dopaminergic neurons. Although the role of α-synuclein in this process is widely documented, its exact function is not clear. The objective of this study was to evaluate the mechanism(s) of dopaminergic degeneration associated with α-synuclein expression in response to Mn exposure and to assess the role of nuclear factor-κB (NF-κB) activation as an intermediary of Mn-induced neurotoxicity. Rat mesencephalic cells (MES 23.5) overexpressing human α-synuclein show enhanced susceptibility to Mn exposure as evidenced by increased apoptosis and NF-κB nuclear translocation. Pretreatment with antioxidants and the p38 mitogen-activated protein kinase (MAPK) inhibitor SB239063 significantly diminished NF-κB activation, supporting a role for oxidative stress and p38 MAPK in Mn-induced NF-κB activation. In addition, increased nitric oxide generation was evident during NF-κB activation, which was blocked by NF-κB (SN50) and MAPK inhibitors. Mn-induced cell death was attenuated by SN-50 and specific nitric oxide synthase (NOS) inhibitor (1400W); corroborating NOS activation is mediated through NF-κB in the mechanism of cell death. These data indicate that the transcription factor NF-κB, p38 MAPK, and apoptotic signaling cascades are activated by Mn in human α-synuclein-overexpressing cells. Thus, α-synuclein may facilitate Mn-induced neurotoxicity, and along with NF-κB, it may play a role in dopaminergic cell death.

Emerging contaminants: presentations at the 2009 Toxicology and Risk Assessment Conference.

A session entitled "Emerging Contaminants" was held in April 2009 in Cincinnati, OH at the 2009 Toxicology and Risk Assessment Conference. The purpose of the session was to share information on both programmatic and technical aspects associated with emerging contaminants. Emerging contaminants are chemicals or materials that are characterized by a perceived or real threat to human health or environment, a lack of published health standards or an evolving standard. A contaminant may also be "emerging" because of the discovery of a new source, a new pathway to humans, or a new detection method or technology. The session included five speakers representing the Department of Defense (DoD), the Environmental Protection Agency (EPA), and each of the military services. The DoD created the Emerging Contaminant Directorate to proactively address environmental, health, and safety concerns associated with emerging contaminants. This session described the scan-watch-action list process, impact assessment methodology, and integrated risk management concept that DoD has implemented to manage emerging contaminants. EPA presented emerging trends in health risk assessment. Researchers made technical presentations on the status of some emerging contaminates in the assessment process (i.e. manganese, RDX, and naphthalene).

BNIP3 up-regulation and mitochondrial dysfunction in manganese-induced neurotoxicity.

The central nervous system (CNS) appears to be the critical target of manganese (Mn), and neurotoxicity has been the focus of most of the health effects of manganese. In brain, the mechanism underlying the Mn-induced cell death is not clear. We have previously demonstrated that NFkappabeta induction and the activation of nitric oxide synthase through reactive oxygen species (ROS) represent a proximate mechanism for Mn-induced neurotoxicity. In this study, an immortalized dopaminergic cells were used to characterize the cell death signaling cascade activated by manganese. Exposure to Mn resulted in a time and concentration-related loss of cell viability as observed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and live/dead cell assay. Mn increased BNIP3 expression within 3h and continued to increase up to 24h exposure followed by a concentration-related apoptotic death as determined by TUNEL. Further, Mn treatment resulted in accumulation of reactive oxygen species and mitochondrial dysfunction with loss of mitochondrial membrane potential and release of cytochrome c. Antioxidants significantly reduced Mn-induced BNIP3 expression and attenuated cell death, demonstrating the role of oxidative stress in BNIP3 induction. Blocking BNIP3 up-regulation with a transcription or a translational inhibitor reduced the response to manganese. Cell death by manganese was reduced in the presence of CsA (PT pore inhibitor). In addition, knockdown of BNIP3 by small interfering RNA (siRNA) improved mitochondrial recovery and reduced neuronal cell loss suggesting that constitutive expression of BNIP3 plays a role in Mn-induced neurotoxicity by regulating mitochondrial functions. These findings indicate a potential detrimental role of BNIP3 in manganese-induced neuronal cell death.

Nineteen-year experience of paediatric renal transplantation in Singapore

<p><b>INTRODUCTION</b>Renal transplantation is the treatment of choice for children with end-stage renal failure (ESRF). The paediatric renal transplant programme in Singapore was initiated in 1989. This study aimed to examine our outcomes over the 19-year period from 1989 to 2007.</p><p><b>MATERIALS AND METHODS</b>A total of 38 renal transplants were performed at our centre. Another 4 patients with overseas transplants who returned within 3 weeks post-transplant were included. The proportion of living donor (LD) transplants was 61.9%. Structural abnormalities and glomerulopathies were the most common aetiologies comprising 33% each. Median age at transplant was 13.9 years and median waiting time was 2.2 years. LD transplant recipients were younger and had a shorter waiting time than deceased donor (DD) recipients.</p><p><b>RESULTS</b>Overall patient survival rates were 95%, 92%, 86% and 86% at 1, 5, 10 and 15 years, respectively. There were 4 deaths, of which 3 were due to infections. Graft survival rates at 1, 5, 10 and 15 years for LD and DD transplants were 100%, 89.5%, 67.3%, 67.3% and 80.8%, 56.5%, 42.2%, 28.3% respectively, and were significantly higher in LD transplants. The main cause of graft loss was rejection following non-adherence. Multivariate analysis showed male gender, late acute rejections and acute tubular necrosis as predictors of graft failure. There was a high incidence of early bacterial infections (42.9%) and cytomegalovirus disease (16.7%).</p><p><b>CONCLUSION</b>Our graft survival rates for LD transplants were comparable to North American rates, although our DD transplant rates were slightly worse, probably a reflection of the prevailing transplant policies.</p>

Sialoblastoma.

Sialoblastoma should be considered in the diagnosis of a perinatal neck swelling that appears to be contiguous with the submandibular salivary gland. These are exceedingly rare perinatal salivary tumors of epithelial origin with variable biologic behavior including the potential for local and systemic recurrence. We report the case of a 3-month-old boy who presented with a submandibular swelling of insidious onset that was initially thought to be a lymph node enlargement.

Recurrent acute thyroid swellings because of pyriform sinus fistula.

Anterior neck abscesses are not rare; but their origin from within the usually infection-resistant thyroid gland is not thought of in the first instance. We encountered 3 patients with differing presentations (tender nodule over anterior neck, recurrent abscess overlying the thyroid gland, and nonhealing fistula with inflammation of the anterior neck). These were caused by persistent embryological communication from the pyriform sinus to the thyroid gland to the left lobe. Excluding the first patient in whom an abnormal communication with oropharynx was suspected when actinomyces was detected in the aspiration cytology of a thyroid nodule, the other 2 patients underwent drainage as for any neck abscess. Fistulous communication was confirmed on barium swallow (in 2 patients) or computed tomographic scan (in 1 patient). En bloc excision of the affected thyroid along with the fistulous tract was performed in all patients. Long-term follow-up confirmed a cure.

Interaction of cyanide and nitric oxide with cytochrome c oxidase: implications for acute cyanide toxicity.

Acute cyanide toxicity is attributed to inhibition of cytochrome c oxidase (CcOX), the oxygen-reducing component of mitochondrial electron transport; however, the mitochondrial action of cyanide is complex and not completely understood. State-3 oxygen consumption and CcOX activity were studied in rat N27 mesencephalic cells to examine the functional interaction of cyanide and nitric oxide (NO). KCN produced a concentration-dependent inhibition of cellular respiration. Cyanide's median inhibitory concentration (IC50) of oxygen consumption (13.2 +/- 1.8microM) was higher than the CcOX IC50 (7.2 +/- 0.1microM). Based on respiratory threshold analysis, 60% inhibition of CcOX was necessary before oxygen consumption was decreased. Addition of high levels of exogenous NO (100microM S-nitroso-N-acetyl-DL-penicillamine) attenuated cyanide inhibition of both respiration and CcOX. On the other hand, when endogenous NO generation was blocked by an NOS inhibitor (N(omega)-monomethyl-L-arginine ester), the cyanide IC50 for both respiration and CcOX increased to 59.6 +/- 0.9microM and 102 +/- 10microM, respectively, thus showing constitutive, low-level NO production enhanced cyanide inhibition. Laser scanning cytometry showed that cyanide elevated mitochondrial NO, which then was available to interact with CcOX to enhance the inhibition. It is concluded that the rapid, potent action of cyanide is due in part to mitochondrial generation of NO, which enhances inhibition of CcOX. At low mitochondrial oxygen tensions, the cyanide-NO interaction would be increased. Also, the antidotal action of sodium nitrite is partly explained by generation of high mitochondrial levels of NO, which antagonizes the CcOX inhibition.

Impact of new legislation on presumed consent on organ donation on liver transplant in Singapore: a preliminary analysis.

Demand for the liver organ for transplantation vastly exceeded the availability of deceased donor organs. A new law, the revised Human Organ Transplant Act (HOTA), was implemented in Singapore in July 2004, which allowed for recovering four organs, including liver, for transplant unless the deceased give objection prior to their demise. We set to study the impact of the revised legislation by comparing the number of potential suitable donors, liver recovery surgery, and liver transplants two years before and one year after the implementation. There was no change in the number of suitable donors, but there was an increase in the number of liver recovery surgeries and liver transplantation, and a lower refusal rate among suitable donors. Although the revised legislation helped improve the availability of deceased donor organs moderately, other nonlegislative, supplementary measures are needed to further improve the low organ donation rate.

PPARalpha-mediated upregulation of uncoupling protein-2 switches cyanide-induced apoptosis to necrosis in primary cortical cells.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a member of the nuclear factor PPAR family that regulates a variety of cellular functions, including lipid metabolism, cellular oxidative stress defense, and inflammatory responses. Based on the report that Wy14,643, a PPARalpha agonist, can upregulate uncoupling protein-2 (UCP-2), this study was conducted in primary cortical cells to determine if PPARalpha activation enhances cyanide-induced neurotoxicity through changes in the level of UCP-2. PCR and Western blot analysis showed that Wy14,643 upregulated UCP-2 transcriptionally over a 12-h period. This response was mediated by PPARalpha since it was blocked by MK886, a selective PPARalpha antagonist. The effect of UCP-2 upregulation on the cytotoxic response to cyanide was quantitated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (apoptosis) and propidium iodide staining (necrosis). Wy14,643 switched the mode of cyanide-induced cell death from apoptosis to necrosis. Cell death was preceded by marked mitochondrial dysfunction, as reflected by depletion of ATP and reduction of the mitochondrial membrane potential (DeltaPsim). Knock down of UCP-2 expression by RNA interference blocked the Wy14,643-mediated enhancement of cyanide-induced mitochondrial dysfunction and the switch of the cell death mode, thus confirming that the response was mediated by upregulation of UCP-2. This study shows that PPARalpha activation can upregulate UCP-2 expression, which in turn enhances cyanide-induced necrotic cell death through an increase of mitochondrial dysfunction.

Population pharmacokinetics of tacrolimus in whole blood and plasma in asian liver transplant patients.

OBJECTIVES: The objectives of this study were to develop population pharmacokinetic models of tacrolimus in an Asian population with whole blood and plasma drug concentration data, to compare the variability of the pharmacokinetic parameters in these two matrices and to search for the main patient characteristics that explain the variability in pharmacokinetic parameters. STUDY DESIGN: Prospective pharmacokinetic assessment followed by model fitting. PATIENTS: Whole blood samples from 31 liver transplant patients in a local hospital receiving oral tacrolimus as part of their immunosuppressive therapy were assessed. Plasma samples from 29 of the 31 patients were also evaluated. Concentrations of tacrolimus in whole blood and plasma were determined by an electrospray high-performance liquid chromatography with tandem mass spectrometry. Two hundred and thirteen whole blood and 157 plasma tacrolimus concentrations were used for building two nonlinear mixed-effects population models to describe the disposition of tacrolimus in whole blood and plasma, respectively. Covariates that were investigated included demographic characteristics, biological markers of liver and renal functions, corticosteroid dose and haematological parameter. RESULTS: A one-compartment model was used to describe the whole blood and plasma concentration-time data of tacrolimus after oral administration. For the whole blood population model, the population estimates of the first-order absorption rate constant (k(a)), apparent clearance based on whole blood concentration after oral administration (CL(B)/F) and apparent volume of distribution based on whole blood concentrations after oral administration (V(d,B)/F) were 2.08h(-1), 14.1 L/h and 217L, respectively. The coefficient of variations (CVs) of interpatient variabilities in CL(B)/F and V(d,B)/F were 65.7% and 63.8%, respectively. Bodyweight, liver and renal function influenced CL(B)/F, while height and haematocrit influenced V(d,B)/F. The residual (unexplained) variability was 34.8%. For the plasma population model, the population estimates of the k(a), apparent clearance based on plasma concentrations after oral administration (CL(P)/F) and apparent volume of distribution based on plasma concentrations after oral administration (V(d,P)/F) were 5.21h(-1), 537 L/h and 563L, respectively. The CVs of interpatient variabilities in CL(P)/F and V(d,P)/F were 96.0% and 105.4%, respectively. Bodyweight was found to influence CL(P)/F, while the erythrocyte-to-plasma concentration ratio influenced V(d,P)/F. The residual (unexplained) variability was 49.8% at the mean plasma concentration of 1.1 ng/mL. CONCLUSIONS: Whole blood and plasma population pharmacokinetic models of tacrolimus in Asian adult and paediatric liver transplant patients were developed using prospective data in a clinical setting. This has identified and quantified sources of interindividual variability in CL(B)/F, V(d,B)/F, CL(P)/F and V(d,P)/F of tacrolimus in Asian liver transplant patients. Information derived from the whole blood population model may subsequently be used by clinicians for dosage individualisation through Bayesian forecasting.

Sclerosing encapsulating peritonitis in a child secondary to peritoneal dialysis.

Sclerosing encapsulating peritonitis, or "abdominal cocoon," is a rare but serious complication of continuous ambulatory peritoneal dialysis. It is characterized by the diffuse appearance of marked sclerotic thickening of the peritoneal membrane resulting in intestinal obstruction. A 14-year-old adolescent boy with a history of end-stage renal failure on continuous ambulatory peritoneal dialysis presented with symptoms of acute intestinal obstruction. A computed tomography scan of the abdomen revealed distended small bowel loops clustered and displaced to the right upper quadrant. The overlying peritoneum was markedly thickened and calcified. Laparotomy confirmed the diagnosis of sclerosing encapsulating peritonitis and the patient was treated with excision of the fibrocollagenous membrane. Postoperatively, he had prolonged ileus requiring parenteral nutritional support and peritoneal dialysis was restarted on postoperative day 10. A high degree of cognizance is needed to facilitate diagnosis and treatment of this uncommon and potentially life-threatening condition.

Up-regulation of uncoupling protein 2 by cyanide is linked with cytotoxicity in mesencephalic cells.

Uncoupling protein 2 (UCP-2) regulates mitochondrial function by increasing proton leak across the inner membrane to dissociate respiration from ATP synthesis and reduce reactive oxygen species generation. A number of studies have shown that UCP-2 expression protects cells from oxidative stress mediated injuries. In the current study, we show UCP-2-mediated reduction in mitochondrial function contributes to the mitochondrial dysfunction and the necrotic death of primary cultured mesencephalic cells (MCs) after exposure to cyanide, a complex IV inhibitor. The necrotic cell death was directly related to the level of mitochondrial dysfunction, as shown by reduction in ATP levels and decreased mitochondrial membrane potential. Treatment with cyanide for 6 h or longer upregulated UCP-2 expression. Blockade of up-regulation with a transcription or a translational inhibitor reduced the response to cyanide. Knockdown with RNAi or transfection with a UCP-2 dominant-negative interfering mutant reduced the cyanide-induced mitochondrial dysfunction and cell death, showing that constitutive expression of UCP-2 plays a role in the response to cyanide. Overexpression of UCP-2 by transfection with human full-length cDNA potentiated the cyanide toxicity. These findings indicate that UCP-2 can serve as a regulator of mitochondria-mediated necrotic cell death, in which enhanced expression can increase the vulnerability of primary MCs to injury due to complex IV-mediated inhibition by cyanide.

Caspase inhibition switches the mode of cell death induced by cyanide by enhancing reactive oxygen species generation and PARP-1 activation.

Execution of apoptosis can involve activation of the caspase family of proteases. Recent studies show that caspase inhibition can switch the morphology of cell death from apoptotic to necrotic without altering the level of death among cell populations. In the present study, the effect of caspase inhibition on cortical (CX) cell death induced by cyanide was investigated. In primary cultured CX cells exposed to cyanide (400 microM), death was primarily apoptotic as indicated by positive TUNEL staining. Reactive oxygen species (ROS) generation and subsequent caspase activation mediated the apoptosis. Inhibition of the caspase cascade with zVAD-fmk switched the apoptotic response to necrotic cell death, as assessed by increased cellular efflux of LDH and propidium iodide uptake by the cells. The change in death mode was accompanied by a marked increase in poly (ADP-ribose) polymerase-1 (PARP-1) activity, reactive oxygen species (ROS) generation, a reduction in the mitochondrial membrane potential (Delta psi(m)), and reduced cellular ATP. Prior treatment of cells with 3-aminobenzamide (3-AB), a PARP-1 inhibitor, prevented the cells from undergoing necrosis and preserved intracellular ATP levels. These findings indicate that apoptosis and necrosis share common initiation pathways and caspase inhibition can switch the apoptotic response to necrosis. Inhibition of PARP-1 preserves cellular ATP levels and in turn blocks execution of the necrotic death pathway.

Calcineurin-mediated Bad translocation regulates cyanide-induced neuronal apoptosis.

In cyanide-induced apoptosis, an increase in cytosolic free Ca2+ and generation of reactive oxygen species are initiation stimuli for apoptotic cell death. Previous studies have shown that cyanide-stimulated translocation of Bax (Bcl-associated X protein) to mitochondria is linked with release of cytochrome c and subsequent activation of a caspase cascade [Shou, Li, Prabhakaran, Borowitz and Isom (2003) Toxicol. Sci. 75, 99-107]. In the present study, the relationship of the cyanide-induced increase in cytosolic free Ca2+ to activation of Bad ( Bcl-2/Bcl-X(L)- antagonist, causing cell death) was determined in cortical cells. Bad is a Ca2+-sensitive pro-apoptotic Bcl-2 protein, which on activation translocates from cytosol to mitochondria to initiate cytochrome c release. In cultured primary cortical cells, cyanide produced a concentration- and time-dependent translocation of Bad from cytosol to mitochondria. Translocation occurred early in the apoptotic response, since mitochondrial Bad was detected within 1 h of cyanide treatment. Mitochondrial levels of the protein continued to increase up to 12 h post-cyanide exposure. Concurrent with Bad translocation, a Ca2+-sensitive increase in cellular calcineurin activity was observed. Increased cytosolic Ca2+ and calcineurin activation stimulated Bad translocation since BAPTA [bis-(o-aminophenoxy)ethane-N, N, N', N'-tetra-acetic acid], an intracellular Ca2+ chelator, and cyclosporin A, a calcineurin inhibitor, significantly reduced translocation. BAPTA also blocked release of cytochrome c from mitochondria as well as apoptosis. Furthermore, treatment of cells with the calcineurin inhibitors cyclosporin A or FK506 blocked the apoptotic response, linking calcineurin activation and the subsequent translocation of Bad to cell death. These observations show that by inducing a rapid increase in cytosolic free Ca2+, cyanide can partially initiate the apoptotic cascade through a calcineurin-mediated translocation of Bad to mitochondria.