Multiple Functions and Regulation of Mammalian Peroxiredoxins.

Peroxiredoxins (Prxs) constitute a major family of peroxidases, with mammalian cells expressing six Prx isoforms (PrxI to PrxVI). Cells produce hydrogen peroxide (H2O2) at various intracellular locations where it can serve as a signaling molecule. Given that Prxs are abundant and possess a structure that renders the cysteine (Cys) residue at the active site highly sensitive to oxidation by H2O2, the signaling function of this oxidant requires extensive and highly localized regulation. Recent findings on the reversible regulation of PrxI through phosphorylation at the centrosome and on the hyperoxidation of the Cys at the active site of PrxIII in mitochondria are described in this review as examples of such local regulation of H2O2 signaling. Moreover, their high affinity for and sensitivity to oxidation by H2O2 confer on Prxs the ability to serve as sensors and transducers of H2O2 signaling through transfer of their oxidation state to bound effector proteins.

Inactivation of peroxiredoxin I by phosphorylation allows localized H(2)O(2) accumulation for cell signaling.

Despite its toxicity, H(2)O(2) is produced as a signaling molecule that oxidizes critical cysteine residues of effectors such as protein tyrosine phosphatases in response to activation of cell surface receptors. It has remained unclear, however, how H(2)O(2) concentrations above the threshold required to modify effectors are achieved in the presence of the abundant detoxification enzymes peroxiredoxin (Prx) I and II. We now show that PrxI associated with membranes is transiently phosphorylated on tyrosine-194 and thereby inactivated both in cells stimulated via growth factor or immune receptors in vitro and in those at the margin of healing cutaneous wounds in mice. The localized inactivation of PrxI allows for the transient accumulation of H(2)O(2) around membranes, where signaling components are concentrated, while preventing the toxic accumulation of H(2)O(2) elsewhere. In contrast, PrxII was inactivated not by phosphorylation but rather by hyperoxidation of its catalytic cysteine during sustained oxidative stress.

Neighborhood Income Is Associated with Health Care Use in Pediatric Short Bowel Syndrome.

OBJECTIVE: To evaluate associations between neighborhood income and burden of hospitalizations for children with short bowel syndrome (SBS). STUDY DESIGN: We used the Pediatric Health Information System (PHIS) database to evaluate associations between neighborhood income and hospital readmissions, readmissions for central line-associated bloodstream infections (CLABSI), and hospital length of stay (LOS) for patients <18 years with SBS hospitalized between January 1, 2006, and October 1, 2015. We analyzed readmissions with recurrent event analysis and analyzed LOS with linear mixed effects modeling. We used a conceptual model to guide our multivariable analyses, adjusting for race, ethnicity, and insurance status. RESULTS: We included 4289 children with 16 347 hospitalizations from 43 institutions. Fifty-seven percent of the children were male, 21% were Black, 19% were Hispanic, and 67% had public insurance. In univariable analysis, children from low-income neighborhoods had a 38% increased risk for all-cause hospitalizations (rate ratio [RR] 1.38, 95% CI 1.10-1.72, P = .01), an 83% increased risk for CLABSI hospitalizations (RR 1.83, 95% CI 1.37-2.44, P < .001), and increased hospital LOS (ß 0.15, 95% CI 0.01-0.29, P = .04). In multivariable analysis, the association between low-income neighborhoods and elevated risk for CLABSI hospitalizations persisted (RR 1.70, 95% CI 1.23-2.35, P < .01, respectively). CONCLUSIONS: Children with SBS from low-income neighborhoods are at increased risk for hospitalizations due to CLABSI. Examination of specific household- and neighborhood-level factors contributing to this disparity may inform equity-based interventions.

The influence of neighborhood income on healthcare utilization in pediatric liver transplant.

OBJECTIVES: Neighborhood contextual factors are associated with liver transplant outcomes. We analyzed associations between neighborhood-level socioeconomic status and healthcare utilization for pediatric liver transplant recipients. METHODS: We merged the Pediatric Health Information System and Scientific Registry of Transplant Recipients databases and included liver transplant recipients ≤21 years hospitalized between January 2004 and May 2022. Outcomes were annual inpatient bed-days, risk of hospitalizations, and risk of liver biopsies. The primary exposure was zip code-based neighborhood income at transplant. We applied causal inference for variable selection in multivariable analysis. We modeled annual inpatient bed-days with mixed-effect zero-inflated Poisson regression, and rates of hospitalization and liver biopsy with a Cox-type proportional rate model. RESULTS: We included 1006 participants from 29 institutions. Children from low-income neighborhoods were more likely to be publicly insured (67% vs. 46%), Black (20% vs. 12%), Hispanic (30% vs. 17%), and have higher model for end-stage liver disease/pediatric end-stage liver disease model scores at transplant (17 vs. 13) than the remaining cohort. We found no differences in inpatient bed-days or rates of hospitalization across neighborhood groups. In univariable analysis, low-income neighborhoods were associated with increased rates of liver biopsy (rate ratio [RR]: 1.57, 95% confidence interval [CI]: 1.04-2.34, p = 0.03). These findings persisted after adjusting for insurance, race, and ethnicity (RR: 1.86, 95% CI: 1.23-2.83, p < 0.01). CONCLUSIONS: Children from low-income neighborhoods undergo more liver biopsies than other children. These procedures are invasive and potentially preventable. In addition to improving outcomes, interventions to mitigate health inequities among liver transplant recipients may reduce resource utilization.

Circadian Oscillation of Sulfiredoxin in the Mitochondria.

Hydrogen peroxide (H2O2) released from mitochondria regulates various cell signaling pathways. Given that H2O2-eliminating enzymes such as peroxiredoxin III (PrxIII) are abundant in mitochondria, however, it has remained unknown how such release can occur. Active PrxIII-SH undergoes reversible inactivation via hyperoxidation to PrxIII-SO2, which is then reduced by sulfiredoxin. We now show that the amounts of PrxIII-SO2 and sulfiredoxin undergo antiphasic circadian oscillation in the mitochondria of specific tissues of mice maintained under normal conditions. Cytosolic sulfiredoxin was found to be imported into the mitochondria via a mechanism that requires formation of a disulfide-linked complex with heat shock protein 90, which is promoted by H2O2 released from mitochondria. The imported sulfiredoxin is degraded by Lon in a manner dependent on PrxIII hyperoxidation state. The coordinated import and degradation of sulfiredoxin provide the basis for sulfiredoxin oscillation and consequent PrxIII-SO2 oscillation in mitochondria and likely result in an oscillatory H2O2 release.

Caregiver perspectives on the everyday medical and social needs of long-term pediatric liver transplant patients.

Using in-depth interviews, we sought to characterize the everyday medical and social needs of pediatric liver transplant caregivers to inform the future design of solutions to improve care processes. Participants (parents/caregivers of pediatric liver transplant recipients) completed a survey (assessing socioeconomic status, economic hardship, health literacy, and social isolation). We then asked participants to undergo a 60-min virtual, semistructured qualitative interview to understand the everyday medical and social needs of the caregiver and their household. We intentionally oversampled caregivers who reported a social or economic hardship on the survey. Transcripts were analyzed using thematic analysis and organized around the Capability, Opportunity, Motivation-Behavior model. A total of 18 caregivers participated. Of the participants, 50% reported some form of financial strain, and about half had less than 4 years of college education. Caregivers had high motivation and capability in executing transplant-related tasks but identified several opportunities for improving care. Caregivers perceived the health system to lack capability in identifying and intervening on specific family social needs. Caregiver interviews revealed multiple areas in which family supports could be strengthened, including (1) managing indirect costs of prolonged hospitalizations (e.g., food, parking), (2) communicating with employers to support families' needs, (3) coordinating care across hospital departments, and (4) clarifying care team roles in helping families reduce both medical and social barriers. This study highlights the caregiver perspective on barriers and facilitators to posttransplant care. Future work should identify whether these themes are present across transplant centers. Caregiver perspectives should help inform future interventions aimed at improving long-term outcomes for children after liver transplantation.

Non-operative management of biliary complications after Liver Transplantation in pediatric patients: A 30-year experience.

BACKGROUND: To evaluate the efficacy of percutaneous and endoscopic therapeutic interventions for biliary strictures and leaks following LT in children. METHODS: Retrospective analysis of 49 consecutive pediatric liver transplant recipients (27 girls, 22 boys, mean age at transplant 3.9 years) treated at our institution from 1989 to 2019 for biliary leak and/or biliary stricture was performed. Minimally invasive approach was considered clinically successful if it resulted in patency of the narrowed biliary segment and/or correction of the biliary leak. RESULTS: Forty-two patients had a stricture at the biliary anastomosis; seven had a biliary leak. After an average 13.8 years of follow-up, long-term clinical success with minimally invasive treatment (no surgery or re-transplant) was achieved for 24 children (57%) with biliary stricture and 4 (57%) with biliary leaks. Eight patients required re-transplant; however, only one was due to failure of both percutaneous and surgical management. For biliary strictures, failure of non-surgical management was associated with younger age at stricture diagnosis (p < .02). CONCLUSIONS: Percutaneous and endoscopic management of biliary strictures and leaks after LT in children is associated with a durable result in >50% of children.

A catalytic career: Studies spanning glutamine synthetase, phospholipase C, peroxiredoxin, and the intracellular messenger role of hydrogen peroxide.

I learned biochemistry from P. Boon Chock and Earl Stadtman while working on the regulation of Escherichia coli glutamine synthetase as a postdoctoral fellow at the National Institutes of Health. After becoming a tenured scientist at the same institute, my group discovered, purified, and cloned the first three prototypical members of the phospholipase C family and uncovered the mechanisms by which various cell-surface receptors activate these enzymes to generate diacylglycerol and inositol 1,4,5-trisphosphate. We also discovered the family of peroxiredoxin (Prx) enzymes that catalyze the reduction of H2O2, and we established that mammalian cells express six Prx isoforms that not only protect against oxidative damage but also mediate cell signaling by modulating intracellular H2O2 levels. To validate the signaling role of H2O2, we showed that epidermal growth factor induces a transient increase in intracellular H2O2 levels, and the essential cysteine residue of protein-tyrosine phosphatases is a target for specific and reversible oxidation by the H2O2 produced in such cells. These observations led to a new paradigm in receptor signaling, in which protein tyrosine phosphorylation is achieved not via activation of receptor tyrosine kinases alone but also through concurrent inhibition of protein-tyrosine phosphatases by H2O2 Our studies revealed that Prx isozymes are extensively regulated via phosphorylation as well as by hyperoxidation of the active-site cysteine to cysteine sulfinic acid, with the reverse reaction being catalyzed by sulfiredoxin. This reversible hyperoxidation of Prx was further shown to constitute a universal marker for circadian rhythms in all domains of life.

Factors Associated With Frequent Opioid Use in Children With Acute Recurrent and Chronic Pancreatitis.

OBJECTIVES: The aim of the study was to understand the association of frequent opioid use with disease phenotype and pain pattern and burden in children and adolescents with acute recurrent (ARP) or chronic pancreatitis (CP). METHODS: Cross-sectional study of children <19 years with ARP or CP, at enrollment into the INSPPIRE cohort. We categorized patients as opioid "frequent use" (daily/weekly) or "nonfrequent use" (monthly or less, or no opioids), based on patient and parent self-report. RESULTS: Of 427 children with ARP or CP, 17% reported frequent opioid use. More children with CP (65%) reported frequent opioid use than with ARP (41%, P = 0.0002). In multivariate analysis, frequent opioid use was associated with older age at diagnosis (odds ratio [OR] 1.67 per 5 years, 95% confidence interval [CI] 1.13-2.47, P = 0.01), exocrine insufficiency (OR 2.44, 95% CI 1.13-5.24, P = 0.02), constant/severe pain (OR 4.14, 95% CI 2.06-8.34, P < 0.0001), and higher average pain impact score across all 6 functional domains (OR 1.62 per 1-point increase, 95% CI 1.28-2.06, P < 0.0001). Children with frequent opioid use also reported more missed school days, hospitalizations, and emergency room visits in the past year than children with no frequent use (P < 0.0002 for each). Participants in the US West and Midwest accounted for 83% of frequent opioid users but only 56% of the total cohort. CONCLUSIONS: In children with CP or ARP, frequent opioid use is associated with constant pain, more healthcare use, and higher levels of pain interference with functioning. Longitudinal and prospective research is needed to identify risk factors for frequent opioid use and to evaluate nonopioid interventions for reducing pain and disability in these children.

Feedback control of adrenal steroidogenesis via H2O2-dependent, reversible inactivation of peroxiredoxin III in mitochondria.

Certain members of the peroxiredoxin (Prx) family undergo inactivation through hyperoxidation of the catalytic cysteine to sulfinic acid during catalysis and are reactivated by sulfiredoxin; however, the physiological significance of this reversible regulatory process is unclear. We now show that PrxIII in mouse adrenal cortex is inactivated by H(2)O(2) produced by cytochrome P450 enzymes during corticosterone production stimulated by adrenocorticotropic hormone. Inactivation of PrxIII triggers a sequence of events including accumulation of H(2)O(2), activation of p38 mitogen-activated protein kinase, suppression of steroidogenic acute regulatory protein synthesis, and inhibition of steroidogenesis. Interestingly, levels of inactivated PrxIII, activated p38, and sulfiredoxin display circadian oscillations. Steroidogenic tissue-specific ablation of sulfiredoxin in mice resulted in the persistent accumulation of inactive PrxIII and suppression of the adrenal circadian rhythm of corticosterone production. The coupling of CYP11B1 activity to PrxIII inactivation provides a feedback regulatory mechanism for steroidogenesis that functions independently of the hypothalamic-pituitary-adrenal axis.

Impact of the Pediatric End-Stage Liver Disease (PELD) growth failure thresholds on mortality among pediatric liver transplant candidates.

The Pediatric End-Stage Liver Disease (PELD) score is intended to determine priority for children awaiting liver transplantation. This study examines the impact of PELD's incorporation of "growth failure" as a threshold variable, defined as having weight or height <2 standard deviations below the age and gender norm (z-score <2). First, we demonstrate the "growth failure gap" created by PELD's current calculation methods, in which children have z-scores <2 but do not meet PELD's growth failure criteria and thus lose 6-7 PELD points. Second, we utilized United Network for Organ Sharing (UNOS) data to investigate the impact of this "growth failure gap." Among 3291 pediatric liver transplant candidates, 26% met PELD-defined growth failure, and 17% fell in the growth failure gap. Children in the growth failure gap had a higher risk of waitlist mortality than those without growth failure (adjusted subhazard ratio [SHR] 1.78, 95% confidence interval [95% CI] 1.05-3.02, P = .03). They also had a higher risk of posttransplant mortality (adjusted HR 1.55, 95% CI 1.03-2.32, P = .03). For children without PELD exception points (n = 1291), waitlist mortality risk nearly tripled for those in the gap (SHR 2.89, 95% CI 1.39-6.01, P = .005). Current methods for determining growth failure in PELD disadvantage candidates arbitrarily and increase their waitlist mortality risk. PELD should be revised to correct this disparity.

Pancreas Divisum in Pediatric Acute Recurrent and Chronic Pancreatitis: Report From INSPPIRE.

INTRODUCTION: The significance of pancreas divisum (PD) as a risk factor for pancreatitis is controversial. We analyzed the characteristics of children with PD associated with acute recurrent or chronic pancreatitis to better understand its impact. PATIENTS AND METHODS: We compared children with or without PD in the well-phenotyped INSPPIRE (INternational Study group of Pediatric Pancreatitis: In search for a cuRE) cohort. Differences were analyzed using 2-sample t test or Wilcoxon rank sum test for continuous variables, Pearson χ or Fisher exact test for categorical variables. RESULTS: PD was found in 52 of 359 (14.5%) subjects, a higher prevalence than the general population (∼7%). Females more commonly had PD (71% vs. 55%; P=0.02). Children with PD did not have a higher incidence of mutations in SPINK1, CFTR, CTRC compared with children with no PD. Children with PD were less likely to have PRSS1 mutations (10% vs. 34%; P<0.01) or a family history of pancreatitis (P<0.05), and more likely to have hypertriglyceridemia (11% vs. 3%; P=0.03). Children with PD underwent significantly more endoscopic procedures and pancreatic sphincterotomy. Patients with PD had fewer attacks of acute pancreatitis (P=0.03) and were less likely to develop exocrine pancreatic insufficiency (P=0.01). Therapeutic endoscopic retrograde cholangiopancreatography was considered most helpful if pancreatic duct was impacted with stones (83% helpful). CONCLUSIONS: PD is likely a risk factor for acute recurrent pancreatitis and chronic pancreatitis in children that appears to act independently of genetic risk factors. Patients with PD and stones obstructing the pancreatic duct benefit most from therapeutic endoscopic retrograde cholangiopancreatography.

Center variation in screening for and management of metabolic syndrome in pediatric liver transplant recipients: A survey of SPLIT centers.

INTRODUCTION: PTMS-a clustering of hypertension, dyslipidemia, glucose intolerance/diabetes, and obesity-is increasingly recognized as a contributor to long-term morbidity after transplant. We sought to describe pediatric liver transplant center protocols and provider practices in screening for and managing these conditions. METHODS: Cross-sectional survey of pediatric liver transplant providers from centers that participate in Studies of Liver Transplantation (SPLIT). RESULTS: Of 49 survey respondents from 39 centers, 64% were hepatologists or surgeons, 18% nurses/NPs/PAs, 12% coordinators, and 4% other. All providers felt that pediatric liver transplant recipients should be routinely screened for PTMS components. For each condition, at least 70% felt that the liver transplant team should be primarily responsible for routine screening. For each condition, at least 30% of providers reported that their center had no standardized protocol for screening. For diagnostic evaluation and initial management, >60% of providers reported that their center had no standardized protocol for glucose intolerance/diabetes, dyslipidemia, or obesity. Almost 40% had no standardized workup or initial management protocol for hypertension or chronic kidney disease. Of centers that did have screening or workup protocols, most were based on existing center practice, provider consensus, or informal review of published evidence. Screening tools, treatment steps, and thresholds for referral to another specialist varied widely. CONCLUSIONS: Transplant providers intend to screen for and initiate management of PTMS components in these children, but protocols and practices vary substantially. This highlights opportunities for multi-center collaboration on protocols or interventions to improve screening and management.

Risk Factors for Rapid Progression From Acute Recurrent to Chronic Pancreatitis in Children: Report From INSPPIRE.

OBJECTIVE: The aim of the study was to determine the rate of progression from acute recurrent pancreatitis (ARP) to chronic pancreatitis (CP) in children and assess risk factors. STUDY DESIGN: Data were collected from the INternational Study group of Pediatric Pancreatitis: In search for a cuRE (INSPPIRE) cohort. Kaplan-Meier curves were constructed to calculate duration of progression from initial attack of acute pancreatitis (AP) to CP. Log-rank test was used to compare survival (nonprogression) probability distribution between groups. Cox proportional hazard regression models were fitted to obtain hazard ratio (with 95% confidence interval [CI]) of progression for each risk variable. RESULTS: Of 442 children, 251 had ARP and 191 had CP. The median time of progression from initial attack of AP to CP was 3.79 years. The progression was faster in those ages 6 years or older at the first episode of AP compared to those younger than 6 years (median time to CP: 2.91 vs 4.92 years; P = 0.01). Children with pathogenic PRSS1 variants progressed more rapidly to CP compared to children without PRSS1 variants (median time to CP: 2.52 vs 4.48 years; P = 0.003). Within 6 years after the initial AP attack, cumulative proportion with exocrine pancreatic insufficiency was 18.0% (95% CI: 12.4%, 25.6%); diabetes mellitus was 7.7% (95% CI: 4.2%, 14.1%). CONCLUSIONS: Children with ARP rapidly progress to CP, exocrine pancreatic insufficiency, and diabetes. The progression to CP is faster in children who were 6 years or older at the first episode of AP or with pathogenic PRSS1 variants. The factors that affect the aggressive disease course in childhood warrant further investigation.

Chronic Pancreatitis: Pediatric and Adult Cohorts Show Similarities in Disease Progress Despite Different Risk Factors.

OBJECTIVES: The aim of the present study was to investigate the natural history of chronic pancreatitis (CP); patients in the North American Pancreatitis Study2 (NAPS2, adults) and INternational Study group of Pediatric Pancreatitis: In search for a cuRE (INSPPIRE, pediatric) were compared. METHODS: Demographics, risk factors, disease duration, management and outcomes of 224 children and 1063 adults were compared using appropriate statistical tests for categorical and continuous variables. RESULTS: Alcohol was a risk in 53% of adults and 1% of children (P < 0.0001); tobacco in 50% of adults and 7% of children (P < 0.0001). Obstructive factors were more common in children (29% vs 19% in adults, P = 0.001). Genetic risk factors were found more often in children. Exocrine pancreatic insufficiency was similar (children 26% vs adult 33%, P = 0.107). Diabetes was more common in adults than children (36% vs 4% respectively, P < 0.0001). Median emergency room visits, hospitalizations, and missed days of work/school were similar across the cohorts. As a secondary analysis, NAPS2 subjects with childhood onset (NAPS2-CO) were compared with INSPPIRE subjects. These 2 cohorts were more similar than the total INSPPIRE and NAPS2 cohorts, including for genetic risk factors. The only risk factor significantly more common in the NAPS2-CO cohort compared with the INSPPIRE cohort was alcohol (9% NAPS2-CO vs 1% INSPPIRE cohorts, P = 0.011). CONCLUSIONS: Despite disparity in age of onset, children and adults with CP exhibit similarity in demographics, CP treatment, and pain. Differences between groups in radiographic findings and diabetes prevalence may be related to differences in risk factors associated with disease and length of time of CP.

De novo inflammatory bowel disease after pediatric kidney or liver transplant.

A subset of children who receive a liver and/or kidney transplant develop de novo inflammatory bowel disease-like chronic intestinal inflammation, not explained by infection or medications, following transplant. We have conducted a single-center, retrospective case series describing the unique clinical and histologic features of this IBD-like chronic intestinal inflammation following solid organ transplant. At our center, nine of 327 kidney or liver recipients developed de novo IBD following transplant (six liver, two kidney, one liver-kidney). Most children presented with prolonged hematochezia and diarrhea and were treated with aminosalicylates. At time of diagnosis, five were not currently using mycophenolate mofetil for transplant immunosuppression. Histologic and endoscopic findings at IBD diagnosis included inflammation, ulcerations, granulomas, and chronic colitis. Since diagnosis, no patients have required surgical intervention, or escalation to biologic therapy, nor developed stricturing or perianal disease. In this case series, de novo post-transplant IBD developed in 4% of pediatric liver and/or kidney recipients; however, it often does not fit the classic patterns of Crohn's disease or ulcerative colitis.

Circadian rhythm of hyperoxidized peroxiredoxin II is determined by hemoglobin autoxidation and the 20S proteasome in red blood cells.

The catalytic cysteine of the typical 2-Cys Prx subfamily of peroxiredoxins is occasionally hyperoxidized to cysteine sulfinic acid during the peroxidase catalytic cycle. Sulfinic Prx (Prx-SO2H) is reduced back to the active form of the enzyme by sulfiredoxin. The abundance of Prx-SO2H was recently shown to oscillate with a period of ∼24 h in human red blood cells (RBCs). We have now investigated the molecular mechanism and physiological relevance of such oscillation in mouse RBCs. Poisoning of RBCs with CO abolished Prx-SO2H formation, implicating H2O2 produced from hemoglobin autoxidation in Prx hyperoxidation. RBCs express the closely related PrxI and PrxII isoforms, and analysis of RBCs deficient in either isoform identified PrxII as the hyperoxidized Prx in these cells. Unexpectedly, RBCs from sulfiredoxin-deficient mice also exhibited circadian oscillation of Prx-SO2H. Analysis of the effects of protease inhibitors together with the observation that the purified 20S proteasome degraded PrxII-SO2H selectively over nonhyperoxidized PrxII suggested that the 20S proteasome is responsible for the decay phase of PrxII-SO2H oscillation. About 1% of total PrxII undergoes daily oscillation, resulting in a gradual loss of PrxII during the life span of RBCs. PrxII-SO2H was detected in cytosolic and ghost membrane fractions of RBCs, and the amount of membrane-bound PrxII-SO2H oscillated in a phase opposite to that of total PrxII-SO2H. Our results suggest that membrane association of PrxII-SO2H is a tightly controlled process and might play a role in the tuning of RBC function to environmental changes.

Selective inhibition of the function of tyrosine-phosphorylated STAT3 with a phosphorylation site-specific intrabody.

Signal transducer and activator of transcription 3 (STAT3) is a multifunctional protein that participates in signaling pathways initiated by various growth factors and cytokines. It exists in multiple forms including those phosphorylated on Tyr(705) (pYSTAT3) or Ser(727) (pSSTAT3) as well as the unphosphorylated protein (USTAT3). In addition to the canonical transcriptional regulatory role of pYSTAT3, both USTAT3 and pSSTAT3 function as transcriptional regulators by binding to distinct promoter sites and play signaling roles in the cytosol or mitochondria. The roles of each STAT3 species in different biological processes have not been readily amenable to investigation, however. We have now prepared an intrabody that binds specifically and with high affinity to the tyrosine-phosphorylated site of pYSTAT3. Adenovirus-mediated expression of the intrabody in HepG2 cells as well as mouse liver blocked both the accumulation of pYSTAT3 in the nucleus and the production of acute phase response proteins induced by interleukin-6. Intrabody expression did not affect the overall accumulation of pSSTAT3 induced by interleukin-6 or phorbol 12-myristate 13-acetate (PMA), the PMA-induced expression of the c-Fos gene, or the PMA-induced accumulation of pSSTAT3 specifically in mitochondria. In addition, it had no effect on interleukin-6-induced expression of the gene for IFN regulatory factor 1, a downstream target of STAT1. Our results suggest that the engineered intrabody is able to block specifically the downstream effects of pYSTAT3 without influencing those of pSSTAT3, demonstrating the potential of intrabodies as tools to dissect the cellular functions of specific modified forms of proteins that exist as multiple species.

Improved enteral tolerance following step procedure: systematic literature review and meta-analysis.

Surgical management of children with short bowel syndrome (SBS) changed with the introduction of the serial transverse enteroplasty procedure (STEP). We conducted a systematic review and meta-analysis using MEDLINE and SCOPUS to determine if children with SBS had improved enteral tolerance following STEP. Studies were included if information about a child's pre- and post-STEP enteral tolerance was provided. A random effects meta-analysis provided a summary estimate of the proportion of children with enteral tolerance increase following STEP. From 766 abstracts, seven case series involving 86 children were included. Mean percent tolerance of enteral nutrition improved from 35.1 to 69.5. Sixteen children had no enteral improvement following STEP. A summary estimate showed that 87 % (95 % CI 77-95 %) of children who underwent STEP had an increase in enteral tolerance. Compilation of the literature supports the belief that SBS subjects' enteral tolerance improves following STEP. Enteral nutritional tolerance is a measure of efficacy of STEP and should be presented as a primary or secondary outcome. By standardizing data collection on children undergoing STEP procedure, better determination of nutritional benefit from STEP can be ascertained.