Genetic Susceptibility to Postdiarrheal Hemolytic-Uremic Syndrome After Shiga Toxin-Producing Escherichia coli Infection: A Centers for Disease Control and Prevention FoodNet Study.

Background: Postdiarrheal hemolytic-uremic syndrome (D+HUS) following Shiga toxin-producing Escherichia coli (STEC) infection is a serious condition lacking specific treatment. Host immune dysregulation and genetic susceptibility to complement hyperactivation are implicated in non-STEC-related HUS. However, genetic susceptibility to D+HUS remains largely uncharacterized. Methods: Patients with culture-confirmed STEC diarrhea, identified through the Centers for Disease Control and Prevention FoodNet surveillance system (2007-2012), were serotyped and classified by laboratory and/or clinical criteria as having suspected, probable, or confirmed D+HUS or as controls and underwent genotyping at 200 loci linked to nondiarrheal HUS or similar pathologies. Genetic associations with D+HUS were explored by multivariable regression, with adjustment for known risk factors. Results: Of 641 enrollees with STEC O157:H7, 80 had suspected D+HUS (41 with probable and 32 with confirmed D+HUS). Twelve genes related to cytokine signaling, complement pathways, platelet function, pathogen recognition, iron transport, and endothelial function were associated with D+HUS in multivariable-adjusted analyses (P ≤ .05). Of 12 significant single-nucleotide polymorphisms (SNPs), 5 were associated with all levels of D+HUS (intergenic SNP rs10874639, TFRC rs3804141, EDN1 rs5370, GP1BA rs121908064, and B2M rs16966334), and 7 SNPs (6 non-complement related) were associated with confirmed D+HUS (all P < .05). Conclusions: Polymorphisms in many non-complement-related genes may contribute to D+HUS susceptibility. These results require replication, but they suggest novel therapeutic targets in patients with D+HUS.

The impact of mitochondrial and nuclear DNA variants on late-onset Alzheimer's disease risk.

We investigated the potential contribution of mitochondrial DNA (mtDNA) variants, haplogroups, and polymorphisms in nuclear genes essential for mitochondrial biogenesis and function (PGC-1α TFAM) to late-onset Alzheimer's disease (LOAD) risk. Epistatic interaction analysis was conducted between the studied variables. Our results demonstrate that mtDNA haplogroups and subhaplogroups with putative role in partial uncoupling of oxidative phosphorylation are significantly associated with a decreased LOAD risk (OR <1). Conversely, mtDNA haplogroup H (p = 0.049) and HV cluster (p = 0.018) are significant LOAD risk factors, which was additionally confirmed by meta-analysis (OR = 1.22, OR = 1.25, respectively). Haplogroup K was demonstrated to exert a neutralizing effect on the high risk associated with APOE4+ status (p = 0.014). Further, two synergistic interactions between subhaplogroup H5 and APOE4 status (p = 0.009) and between TFAM rs1937 and APOE4 status (p < 0.001) were detected, influencing LOAD risk. No interaction pointing to a dual mitochondrial-nuclear genome variation effect on LOAD occurrence was identified.

Mitochondrial genomics and CD4 T-cell count recovery after antiretroviral therapy initiation in AIDS clinical trials group study 384.

BACKGROUND: Mitochondrial DNA (mtDNA) variation has been associated with time to progression to AIDS and adverse effects from antiretroviral therapy (ART). In this study, full mitochondrial DNA (mtDNA) sequence data from US-based adult participants in the AIDS Clinical Trials Group study 384 was used to assess associations between mtDNA variants and CD4 T-cell recovery with ART. METHODS: Full mtDNA sequence was determined using chip-based array sequencing. Sequence and CD4 cell count data was available at baseline and after ART initiation for 423 subjects with HIV RNA levels <400 copies per milliliter plasma. The primary outcome was change in CD4 count of ≥100 cells per cubic millimeter from baseline. Analyses were adjusted for baseline age, CD4 cell count, HIV RNA, and naive:memory CD4 cell ratio. RESULTS: Race-stratified analysis of mtDNA variants with a minor allele frequency >1% revealed multiple mtDNA variants marginally associated (P < 0.05 before Bonferroni correction) with CD4 cell recovery. The most significant single nucleotide polymorphism associations were those tagging the African L2 haplogroup, which was associated with a decreased likelihood of ≥100 cells per cubic millimeter CD4 count increase at week 48 in non-Hispanic blacks (adjusted odds ratio = 0.17; 95% confidence interval = 0.06 to 0.53; P = 0.002). CONCLUSIONS: An African mtDNA haplogroup was associated with CD4 cell recovery after ART in this clinical trial population. These initial findings warrant replication and further investigation to confirm the role of mtDNA variation in CD4 cell recovery during ART.

European mitochondrial DNA haplogroups and metabolic changes during antiretroviral therapy in AIDS Clinical Trials Group Study A5142.

BACKGROUND: Mitochondrial DNA (mtDNA) influences metabolic diseases and perhaps antiretroviral therapy (ART) complications. We explored associations between European mtDNA haplogroups and metabolic changes among A5142 participants. METHODS: Seven hundred and fifty-seven ART-naive patients were randomized to one of three class-sparing ART regimens including efavirenz and/or lopinavir/ritonavir with or without nucleoside reverse transcriptase inhibitors (NRTIs). Nonrandomized NRTIs included stavudine, tenofovir, or zidovudine, each with lamivudine. Fasting lipid profiles and whole-body dual-energy X-ray absorptiometry (DEXA) were performed. Nine European mtDNA haplogroups were determined for 231 self-identified non-Hispanic white individuals. Metabolic changes from baseline to 96 weeks were analyzed by haplogroup. RESULTS: Median age was 39 years, 9% were women, and 37, 32, and 30 were randomized to NRTI-containing regimens with either efavirenz or lopinavir/ritonavir, and an NRTI-sparing regimen, respectively. Among NRTI-containing regimens, 51% included zidovudine, 28% tenofovir, and 21% stavudine. Compared with other haplogroups, mtDNA haplogroup I (N = 10) had higher baseline non-HDL cholesterol [160 mg/dl (interquartile range 137-171) vs. 120 mg/dl (104-136); P = 0.005], a decrease in non-HDL cholesterol over 96 weeks [-14% (-20 to 6) vs. +25% (8 to 51); P < 0.001], tended to have more baseline extremity fat, and had more extremity fat loss by DEXA [-13% (-13 to 12) vs. +9% (-13 to 26); P = 0.08] and lipoatrophy (50 vs. 20%; P = 0.04). Haplogroup W (N = 5; all randomized to NRTI-sparing regimens) had the greatest increase in extremity fat [+35.5% (26.8 to 54.9); P = 0.02]. CONCLUSIONS: Lipids and extremity fat were associated with European mtDNA haplogroups in this HIV-infected population. These preliminary results suggest that mitochondrial genomics may influence metabolic parameters before and during ART.

An ethnic-specific polymorphism in the catalytic subunit of glutamate-cysteine ligase impairs the production of glutathione intermediates in vitro.

Glutathione plays a crucial role in free radical scavenging, oxidative injury, and cellular homeostasis. Previously, we identified a non-synonymous polymorphism (P462S) in the gene encoding the catalytic subunit of glutamate-cysteine ligase (GCLC), the rate-limiting enzyme in glutathione biosynthesis. This polymorphism is present only in individuals of African descent. Presently, we report that this ethnic-specific polymorphism (462S) encodes an enzyme with significantly decreased in vitro activity when expressed by either a bacterial or mammalian cell expression system. In addition, overexpression of the 462P wild-type GCLC enzyme results in higher intracellular glutathione concentrations than overexpression of the 462S isoform. We also demonstrate that apoptotically stimulated mammalian cells overexpressing the 462S enzyme have increased caspase activation and increased DNA laddering compared to cells overexpressing the wild-type 462P enzyme. Finally, we genotyped several African and African-descent populations and demonstrate that the 462S polymorphism is in Hardy-Weinberg disequilibrium, with no individuals homozygous for the 462S polymorphism identified. These findings describe a glutathione production pathway polymorphism present in individuals of African descent with significantly decreased in vitro activity.

African mitochondrial DNA subhaplogroups and peripheral neuropathy during antiretroviral therapy.

Susceptibility to peripheral neuropathy during antiretroviral therapy with nucleoside reverse-transcriptase inhibitors was previously associated with a European mitochondrial DNA (mtDNA) haplogroup among non-Hispanic white persons. To determine whether nucleoside reverse-transcriptase inhibitor-associated peripheral neuropathy was related to mtDNA variation in non-Hispanic black persons, we sequenced mtDNA of participants from AIDS Clinical Trials Group study 384. Of 156 non-Hispanic black persons with genomic data, 51 (33%) developed peripheral neuropathy. In a multivariate model, African mtDNA subhaplogroup L1c was an independent predictor of peripheral neuropathy (odds ratio, 3.7 [95% confidence interval, 1.1-12.0]). An African mtDNA subhaplogroup is for the first time implicated in susceptibility to nucleoside reverse-transcriptase inhibitor-associated toxicity.

Mitochondrial transcription factor A variants and the risk of Parkinson's disease.

The mitochondrial transcription factor A (TFAM) has been recently shown to decrease reactive oxygen species (ROS) generation. It is also known that mitochondrial DNA (mtDNA) haplogroups might confer different coupling properties, resulting in different ROS levels. We hypothesized that potentially functional TFAM variants could influence PD risk depending on haplogroup background. To test this we assessed the role of six TFAM variants on PD risk in 326 PD patients and 316 controls, and correlated it with mtDNA haplogroup clusters (HV, JTKU and a putative functionally different group U4U5a1KJ1cJ2, connected previously with partial uncoupling of oxidative phosphorylation). Both genotype and haplotype analysis showed that intronic variant rs2306604 modifies PD risk. Multivariate logistic regression analysis confirmed that rs2306604 G/G genotype is an independent risk factor for PD (OR 1.789, 95% CI 1.162-2.755, p=0.008). There was a borderline interaction between G/G genotype and HV haplogroup (p=0.075). Haplotype analysis showed that all three haplotypes containing rs2306604 allele A occurred at higher frequencies in controls, but only one of them reached statistical significance (chi(2) 4.523, p=0.0334). Conversely, four of five haplotypes containing allele G had higher frequencies in PD group, with no statistical significance.

Cyclosporine A suppresses keratinocyte cell death through MPTP inhibition in a model for skin cancer in organ transplant recipients.

Transplant recipients have an elevated risk of skin cancer, with a 65- to 250-fold increase in squamous cell carcinoma. Usage of the immunosuppressant cyclosporine A (CsA) is associated with the development of skin cancer. We hypothesized that the increased incidence of skin cancer was due to the action of CsA within keratinocyte mitochondria where it can inhibit mitochondrial permeability transition pore (MPTP) opening. Normally, MPTP opening is induced by oxidative stress such as that caused by UV light and leads to cell death, thereby eliminating a cell that has been exposed to genotoxic insult. However, in the presence of CsA, damaged cells may survive and consequently form tumors. To test this hypothesis, we treated keratinocytes with levels of CsA used therapeutically in transplant patients and assessed their viability following UVA-irradiation. CsA prevented cell death by inhibiting MPTP opening, even though the levels of oxidative stress were increased markedly. Nim811, a non-immunosuppressive drug that can block the MPTP had a similar effect while the immunosuppressive drug tacrolimus that does not interact with the mitochondria had no effect. These findings suggest that CsA may promote skin cancer in transplant patients by allowing keratinocyte survival under conditions of increased genotoxic stress.

Personalized medicine: genetic variation and loss of physiologic complexity are associated with mortality in 644 trauma patients.

OBJECTIVE: Personalized medicine merges genetics, physiology, and patient outcome. Loss of physiologic complexity (heart rate [HR] variability) is a bedside biomarker for autonomic nervous system (ANS) dysfunction. We hypothesized that variability in ANS receptor proteins (genetics) and loss of complexity (physiology) are independently associated with mortality in critical illness. SUMMARY BACKGROUND DATA: Decreased HR complexity has been associated with increased mortality and morbidity in trauma and other critically ill populations. Genetic variations in alpha-1A and beta-2 adrenergic receptors (ADRA1A, ADRB2) have been associated with changes in smooth muscle tone in various tissues, and implicated in bronchial hyper-responsiveness, metabolic syndrome, and other disorders. METHODS: A cohort of 644 trauma intensive care unit (ICU) admissions had complexity data and genetic samples. Two ANS receptor polymorphisms (rs1048101, Alpha ADRA1A and rs1042714, Beta ADRB2) were genotyped. Physiologic complexity at various points in the ICU stay was measured using previously-studied integer HR multiscale entropy (MSE) over 6-hour intervals (~21,600 HR data points/interval/patient). Logistic regression assessed the concurrent relationship of genotypes, complexity, and probability of survival, an acuity score incorporating age, injury mechanism/severity, and admission vitals, to risk of death. RESULTS: Of total, 96 patients (15%) died. Nonsurvivors had lower complexity at early, middle, and late portions of ICU stay (median MSE at least 25% less in nonsurvivors, P < 0.001) and lower incidence of the GG ADRB2 genotype (7.5% vs. 18.3%, P < 0.001). In multivariable logistic regression, the GG ADRB2 genotype carried ~3-fold decrease in mortality odds (odd ratio [OR] = 0.33, P = 0.01), independent of significant effects in HR MSE (OR = 0.93, P < 0.001), and probability of survival (OR = 0.22, P < 0.001). CONCLUSIONS: This first study to simultaneously examine ANS genetics, the biomarker complexity, and mortality concludes: (1) ANS genetics and physiologic complexity are independently related to mortality; (2) Genetics and complexity add information over traditional acuity scoring (probability of survival); and (3) Simultaneous assessment of ANS physiology and genetics may yield novel research, diagnostic, and therapeutic opportunities in critical illness.

Genetic variation in the autonomic nervous system affects mortality: a study of 1,095 trauma patients.

BACKGROUND: We have previously demonstrated an unappreciated link between the autonomic nervous system and mortality, heart rate variability, and physiologic complexity. STUDY DESIGN: Genetic variation in adrenergic receptors or key enzymes in catecholamine degradation could be associated with, and potentially explain, autonomic nervous system dysfunction and its impact on mortality after severe trauma. Three genetic polymorphisms critical to the adrenergic pathway were evaluated: beta-2 adrenergic receptor (ADBR2: Q27E), alpha-1a adrenergic receptor (ADRA1A:R347C), and catechol-O-methyl transferase (COMT: V158M). The study population consisted of 1,095 trauma admissions between April 2005 and April 2007. These patients all had genotyping performed using mass spectrometric analysis (Sequenom, Inc). The genetic data were linked with detailed demographic and clinical data. Trauma Related Injury Severity Score (TRISS) probability of survival was used as a composite measure of injury severity, admission physiology, and demographic factors in the multivariate logistic regression analyses of mortality outcomes data. RESULTS: The overall mortality rate for the study population was 14.2% (155 of 1,095). Univariate comparisons of genotypes with mortality revealed a significant association with the ADBR2 polymorphism: CC=15.9%, GC=14.8% and GG=7.6%, p=0.02. The apparently protective ADBR2 GG genotype was seen in 15.5% (170 of 1,095) of the study population. In multivariate analysis, which included adjustment for TRISS, the ADBR2 GG genotype was associated with reduced mortality (odds ratio 0.36, p=0.002). CONCLUSIONS: Genetic variation in the beta-2 adrenergic receptor (ADBR2:Q27E) associated with bronchial constriction appears protective (odds ratio 0.36), perhaps by making the receptor resistant to downregulation. These genetic data support the emerging understanding of critical role of the autonomic nervous system in the response to injury.

Genetic variation in complement component 2 of the classical complement pathway is associated with increased mortality and infection: a study of 627 patients with trauma.

BACKGROUND: Trauma is a disease of inflammation. Complement Component 2 (C2) is a protease involved in activation of complement through the classical pathway and has been implicated in a variety of chronic inflammatory diseases. We hypothesized that genetic variation in C2 (E318D) identifies a high-risk subgroup of patients with trauma reflecting increased mortality and infection (ventilator-associated pneumonia [VAP]). Consequently, genetic variation in C2 may stratify patient risk and illuminate underlying mechanisms for therapeutic intervention. METHODS: DNA samples from 702 patients with trauma were genotyped for C2 E318D and linked with covariates (age: mean 42.8 years, gender: 74% male, ethnicity: 80% white, mechanism: 84% blunt, injury severity score: mean 25.0, admission lactate: mean 3.13 mEq/L) and outcomes: mortality 9.9% and VAP: 18.5%. VAP was defined by quantitative bronchoalveolar lavage (> 10). Multivariate regression analysis determined the relationship of genotype and covariates to risk of death and VAP. However, patients with injury severity score > or = 45 were excluded from the multivariate analysis, as magnitude of injury overwhelms genetics and covariates in determining outcome. RESULTS: Fifty-two patients (8.3%) had the high-risk heterozygous genotype, associated with a significant increase in mortality and VAP. CONCLUSION: In 702 patients with trauma, 8.3% had a high-risk genetic variation in C2 associated with increased mortality (odds ratio = 2.65) and infection (odds ratio = 2.00). This variation: (1) identifies a previously unknown high-risk group for infection and mortality; (2) can be determined at admission; (3) may provide opportunity for early therapeutic intervention; and (4) requires validation in a distinct cohort of patients.

Mitochondrial haplogroup H and Alzheimer's disease--is there a connection?

We evaluated the involvement of the major Caucasian-specific mitochondrial haplogroups (H, I, J, K, T, U, V, W and X), haplogroup clusters (HV, UK, TJ, IWX) and two functional mtSNPs (4216, 4917) in the pathogenesis of Alzheimer's disease (AD) in the Polish population. The frequency distribution of mtDNA haplogroups was non-randomly associated with APOE4 status (chi(2)=73.17, df=1, p<0.0001, OR=5.97, 95% CI 3.90-9.12), however, no haplogroup-specific neutralizing of the APOE4 allele influence was detected. Multivariate analysis suggested the opposite-APOE4 status could modulate the effect of mtDNA haplogroups. We found that HV cluster is significantly associated with the risk of AD, regardless of the APOE4 status (OR=1.59, 95% CI, 1.04-2.44, p=0.032). Contrary to the previous studies, we report no evidence for the involvement of haplogroup U, K, J or T in AD risk. We conclude that further analysis of subtypes of haplogroup H would be necessary to decipher the relation of HV cluster with AD.

Mitochondrial DNA haplogroups and subhaplogroups are associated with Parkinson's disease risk in a Polish PD cohort.

mtDNA common variation is inconsistently reported to modify the risk of Parkinson's disease (PD). We evaluated the impact of the mitochondrial haplogroups, subhaplogroups, coding and non-coding single-nucleotide polymorphisms on PD risk in 241 PD patients and 277 control subjects. After stratification by gender, we found that haplogroup J (OR 0.19; 95% CI 0.069-0.53; P = 0.0014) was associated with a lower PD risk in males. Unexpectedly, subhaplogroup analysis based on the control region (CR) polymorphisms demonstrated that subcluster K1a was more prevalent in healthy controls, while K1c was more frequent in PD patients (P = 0.025 and P = 0.011, respectively; two-tailed Fisher's exact test). Additionally, we confirmed the hypothesis that sublineages (U4 + U5a1 + K+J1c + J2), previously proposed to partially uncouple oxidative phosphorylation (OXPHOS), decrease PD risk (P = 0.027, chi2 with Yates' correction). The putative protective effect of uncoupling mtDNAs against PD might result from decreased production of reactive oxygen species. We propose that stratification into subhaplogroups or by gender could be necessary to reveal the involvement of specific mtDNA sublineages in PD pathogenesis.

Mitochondrial DNA polymorphism A4917G is independently associated with age-related macular degeneration.

The objective of this study was to determine if MTND2*LHON4917G (4917G), a specific non-synonymous polymorphism in the mitochondrial genome previously associated with neurodegenerative phenotypes, is associated with increased risk for age-related macular degeneration (AMD). A preliminary study of 393 individuals (293 cases and 100 controls) ascertained at Vanderbilt revealed an increased occurrence of 4917G in cases compared to controls (15.4% vs.9.0%, p = 0.11). Since there was a significant age difference between cases and controls in this initial analysis, we extended the study by selecting Caucasian pairs matched at the exact age at examination. From the 1547 individuals in the Vanderbilt/Duke AMD population association study (including 157 in the preliminary study), we were able to match 560 (280 cases and 280 unaffected) on exact age at examination. This study population was genotyped for 4917G plus specific AMD-associated nuclear genome polymorphisms in CFH, LOC387715 and ApoE. Following adjustment for the listed nuclear genome polymorphisms, 4917G independently predicts the presence of AMD (OR = 2.16, 95%CI 1.20-3.91, p = 0.01). In conclusion, a specific mitochondrial polymorphism previously implicated in other neurodegenerative phenotypes (4917G) appears to convey risk for AMD independent of recently discovered nuclear DNA polymorphisms.

Hemochromatosis gene polymorphisms, mitochondrial haplogroups, and peripheral lipoatrophy during antiretroviral therapy.

BACKGROUND: Antiretroviral therapy (ART)-associated lipoatrophy involves mitochondrial dysfunction. Iron metabolism impacts mitochondrial function and oxidative stress. Mitochondrial haplogroups and hemochromatosis gene (HFE) polymorphisms have been associated with ART-induced neuropathy. We assessed relationships between these variants and lipoatrophy. METHODS: The AIDS Clinical Trials Group 384 study randomized ART-naive individuals to receive didanosine-stavudine or zidovudine-lamivudine, combined with efavirenz and/or nelfinavir. Substudy A5005s evaluated fat distribution by dual-energy X-ray absorptiometry (DEXA). We characterized HFE polymorphisms 845G>A and 187C>G and European mitochondrial haplogroups in A5005s participants who consented to genetic analyses. RESULTS: Among 96 participants (58% were white, and 10% were female) with baseline and 48 or 64 week DEXA data, the median limb fat change was -8.8% (interquartile range, -28.7% to +15.6%). HFE 187C/G heterozygotes (n = 23) had less limb fat loss than 187C/C homozygotes (n = 71) (+6.1% vs. -12.5%; P = .02) and were less likely to develop lipoatrophy after adjustment for age, sex, race, and ART randomization (odds ratio, 0.31; 95% confidence interval, 0.10-0.95; P = .04). Among non-Hispanic white participants, median limb fat change was +26.1% among 5 participants with mitochondrial haplogroup J, compared with -9.7% among 49 participants with other mitochondrial haplogroups (P = .07). CONCLUSIONS: HFE 187C>G and, possibly, mitochondrial haplogroup J gave relative protection against lipoatrophy during ART in A5005s. These associations should be replicated in other studies.

Specific polymorphic variation in the mitochondrial genome and increased in-hospital mortality after severe trauma.

OBJECTIVE: To determine whether specific genetic variations in the mtDNA that impact energy production and free-radical generation are potential new risk factors for in-hospital mortality after severe trauma. SUMMARY BACKGROUND DATA: Each of the 3 mitochondrial DNA polymorphisms selected for this study (at positions 4216, 10398, 4917) alter the amino acid sequence of different key subunits of Complex I in the electron transport chain. They have been previously implicated in phenotypes involving tissues with high-energy demand, such as the brain and retina. METHODS: Seven hundred forty-five consecutive patients admitted to the trauma intensive care unit at Vanderbilt University Medical Center between April 11, 2005, and February 27, 2006, were potentially eligible for this study. Under an Institutional Review Board-approved protocol (which excluded patients <18 years of age and prisoners), 666 patients had DNA extracted from a blood sample. Detailed demographic and clinical covariates were also obtained (including age, gender, ethnicity, lactate measurements, and injury severity score). A flurogenic 5' nuclease allelic discrimination Taqman assay and the ABI 7900HT Sequence Detection System (v2.1) was used to genotype the T4216C, A10398G, and A4917G polymorphisms. The primary outcome was in-hospital mortality. RESULTS: Multivariate logistic regression analysis revealed that the 4216T allele was a significant independent predictor of in-hospital mortality (OR = 2.63, 95% CI 1.14-6.07, P = 0.02) after adjustment for age, gender, injury severity score, highest lactate level, mechanism of injury, and the 10398 polymorphism. CONCLUSIONS: Variation in the mtDNA, specifically the 4216T allele, appears to increase the risk of in-hospital mortality after severe injury.

Pharmacokinetics and safety of intravenously administered citrulline in children undergoing congenital heart surgery: potential therapy for postoperative pulmonary hypertension.

OBJECTIVE: Pulmonary hypertension may complicate surgical correction of congenital heart defects, resulting in increased morbidity and mortality. We have previously shown that plasma levels of the nitric oxide precursors citrulline and arginine drop precipitously after congenital cardiac surgery and that oral citrulline supplementation may be protective against the development of pulmonary hypertension. In this study, we assessed the safety and pharmacokinetic profile of intravenous citrulline as a potential therapy for postoperative pulmonary hypertension. METHODS: The initial phase of this investigation was a dose-escalation study of intravenously administered citrulline in infants and children undergoing one of five congenital cardiac surgical procedures (phase 1). The primary safety outcome was a 20% drop in mean arterial blood pressure from the baseline pressure recorded after admission to the intensive care unit. Based on our previous work, the target circulating plasma citrulline trough was 80 to 100 micromol/L. Each patient was given two separate doses of citrulline: the first in the operating room immediately after initiation of cardiopulmonary bypass and the second 4 hours later in the pediatric intensive care unit. Stepwise dose escalations included 50 mg/kg, 100 mg/kg, and 150 mg/kg. After model-dependent pharmacokinetic analysis, we enrolled an additional 9 patients (phase 2) in an optimized dosing protocol that replaced the postoperative dose with a continuous infusion of citrulline at 9 mg/(kg.h) for 48 hours postoperatively. RESULTS: The initial stepwise escalation protocol (phase 1) revealed that an intravenous citrulline dose of 150 mg/kg given after initiation of cardiopulmonary bypass yielded a trough level of in the target range of approximately 80 to 100 micromol/L 4 hours later. The postoperative dose revealed that the clearance of intravenously administered citrulline was 0.6 L/(h.kg), with a volume of distribution of 0.9 L/kg and estimated half-life of 60 minutes. Because of the short half-life, we altered the protocol to replace the postoperative dose with a continuous infusion of 9 mg/(kg.h). An additional 9 patients were studied with this continuous infusion protocol (phase 2). Mean plasma citrulline levels were maintained at approximately 125 mumol/L, with a calculated clearance of 0.52 L/(h.kg). None of the 17 patients studied had a 20% drop in mean arterial blood pressure from baseline. CONCLUSIONS: In this first report of the use of intravenous citrulline in humans, we found citrulline to be both safe and well tolerated in infants and young children undergoing congenital cardiac surgery. Because of the rapid clearance, the optimal dosing regimen was identified as an initial bolus of 150 mg/kg given at the initiation of cardiopulmonary bypass, followed 4 hours later by a postoperative infusion of 9 mg/(kg.h) continued up to 48 hours. Using this regimen, plasma arginine, citrulline, and nitric oxide metabolite levels were well maintained. Intravenous citrulline needs to be studied further as a potential therapy for postoperative pulmonary hypertension.

Overexpression of Akt converts radial growth melanoma to vertical growth melanoma.

Melanoma is the cancer with the highest increase in incidence, and transformation of radial growth to vertical growth (i.e., noninvasive to invasive) melanoma is required for invasive disease and metastasis. We have previously shown that p42/p44 MAP kinase is activated in radial growth melanoma, suggesting that further signaling events are required for vertical growth melanoma. The molecular events that accompany this transformation are not well understood. Akt, a signaling molecule downstream of PI3K, was introduced into the radial growth WM35 melanoma in order to test whether Akt overexpression is sufficient to accomplish this transformation. Overexpression of Akt led to upregulation of VEGF, increased production of superoxide ROS, and the switch to a more pronounced glycolytic metabolism. Subcutaneous implantation of WM35 cells overexpressing Akt led to rapidly growing tumors in vivo, while vector control cells did not form tumors. We demonstrated that Akt was associated with malignant transformation of melanoma through at least 2 mechanisms. First, Akt may stabilize cells with extensive mitochondrial DNA mutation, which can generate ROS. Second, Akt can induce expression of the ROS-generating enzyme NOX4. Akt thus serves as a molecular switch that increases angiogenesis and the generation of superoxide, fostering more aggressive tumor behavior. Targeting Akt and ROS may be of therapeutic importance in treatment of advanced melanoma.

Genetic variation in the mitochondrial enzyme carbamyl-phosphate synthetase I predisposes children to increased pulmonary artery pressure following surgical repair of congenital heart defects: a validated genetic association study.

Increased pulmonary artery pressure (PAP) can complicate the postoperative care of children undergoing surgical repair of congenital heart defects. Endogenous NO regulates PAP and is derived from arginine supplied by the urea cycle. The rate-limiting step in the urea cycle is catalyzed by a mitochondrial enzyme, carbamoyl-phosphate synthetase I (CPSI). A well-characterized polymorphism in the gene encoding CPSI (T1405N) has previously been implicated in neonatal pulmonary hypertension. A consecutive modeling cohort of children (N=131) with congenital heart defects requiring surgery was prospectively evaluated to determine key factors associated with increased postoperative PAP, defined as a mean PAP>20 mmHg for at least 1h during the 48h following surgery measured by an indwelling pulmonary artery catheter. Multiple dimensionality reduction (MDR) was used to both internally validate observations and develop optimal two-variable through five-variable models that were tested prospectively in a validation cohort (N=41). Unconditional logistic regression analysis of the modeling cohort revealed that age (OR=0.92, p=0.01), CPSI T1405N genotype (AC vs. AA: OR=4.08, p=0.04, CC vs. AA: OR=5.96, p=0.01), and Down syndrome (OR=5.25, p=0.04) were independent predictors of this complex phenotype. MDR predicted that the best two-variable model consisted of age and CPSI T1405N genotype (p<0.001). This two-variable model correctly predicted 73% of the outcomes from the validation cohort. A five-variable model that added race, gender and Down's syndrome was not significantly better than the two-variable model. In conclusion, the CPSI T1405N genotype appears to be an important new factor in predicting susceptibility to increased PAP following surgical repair of congenital cardiac defects in children.