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OBJECTIVE The treatment of frontoethmoidal meningoencephaloceles (fMECs) in Cambodia was not possible before the development of a program that taught some Khmer surgeons (working at the Children's Surgical Centre in Phnom Penh) how to surgically correct these deformities without any foreign help. The results of that teaching program are discussed in this paper. METHODS Between 2004 and 2009, both local and visiting foreign neurosurgical and craniofacial surgeons (the visitors coming twice a year) worked together to operate on 200 patients, and a report on those cases was published in 2010. In subsequent years (2010-2016), the Khmer surgeons operated on 100 patients without the presence of the visiting surgeons. In this study, the authors compare the second case series with the previously published series and the literature in terms of results and complications. The operations were performed with limited surgical materials and equipment, using a combined bicoronal and transfacial approach in most cases. Most of the patients came from very poor families. RESULTS Organizing the postoperative follow-up of these low-income patients (mean age 12 years) was probably the most challenging part of this teaching program. Nine of the patients were lost to surgical follow-up. In the other cases, cosmetic results were judged by the surgeons as worse than the patient's preoperative appearance in 1 case, poor in 12 cases, average in 27, and good in 51-data that are significantly less encouraging than the results reported by the joint local/visiting teams in 2010 (p = 0.0001). Nevertheless, patients and parents tended to have a better overall opinion about the surgical results (rating the results as good in 84% of the 80 cases in which parent or patient ratings were available). Twenty postoperative complications were observed (the most common being temporary CSF leaks). The rate of immediate postoperative complications directly related to fMEC surgery was less than that in the previous series, but the difference was not statistically significant (20% vs 28.5%, p = 0.58). No death was noted in this case series (in contrast to the previous series). Social questionnaire results confirmed that fMEC correction partially improved the adverse social and educational consequences of fMEC in affected children. CONCLUSIONS In the current state of this program, the local surgeons are able to correct fMECs in their own country, without foreign assistance, with good results in a majority of patients. Such humanitarian teaching programs generally take years to achieve the initial aims.

Hb Kenya is a hemoglobin (Hb) tetramer composed of two normal α- and two non α-globin chains. The latter are the product of a fusion gene in which the 5' end is (A)γ and the 3' end is β. The crossover point is between codon 81 of the (A)γ gene and codon 86 of the β gene. Like the other non α genes, the hybrid protein product ((A)γ81Leu-β86Ala) has 146 amino acids. The purpose of this report is to highlight the laboratory findings of Hb Kenya and to emphasize the pitfalls in misdiagnosis, particularly when associated with another variant such as Hb S [β6(A3)Glu→Val].

Purpose: The primary purpose of this trial was to define and describe the toxicities of oral valproic acid (VPA) at doses required to maintain trough concentrations of 100 to 150 mcg/mL or 150 to 200 mcg/mL in children with refractory solid or central nervous system (CNS) tumors. Secondary objectives included assessment of free and total VPA pharmacokinetics (PKs) and histone acetylation in peripheral blood mononuclear cells (PBMC) at steady state., Patients and Methods: Oral VPA, initially administered twice daily and subsequently three times daily, was continued without interruption to maintain trough concentrations of 100 to 150 mcg/mL. First-dose and steady-state PKs were studied. Histone H3 and H4 acetylation in PBMCs was evaluated using an ELISA technique., Results: Twenty-six children, sixteen of whom were evaluable for toxicity, were enrolled. Dose-limiting somnolence and intratumoral hemorrhage were associated with VPA troughs of 100 to 150 mcg/mL. Therefore, the final cohort of six children received VPA to maintain troughs of 75 to 100 mcg/mL and did not experience any dose-limiting toxicity. First-dose and steady-state VPA PK parameters were similar to values previously reported in children with seizures. Increased PBMC histone acetylation was documented in 50% of patients studied. One confirmed partial response (glioblastoma multiforme) and one minor response (brainstem glioma) were observed., Conclusions: VPA administered three times daily to maintain trough concentrations of 75 to 100 mcg/mL was well tolerated in children with refractory solid or CNS tumors. Histone hyperacetylation in PBMCs was observed in half of the patients at steady state. Future trials combining VPA with chemotherapy and/or radiation therapy should be considered, especially for CNS tumors., (©2010 AACR.)

Background: The clinical measures of gastric residuals and abdominal distention are often used to guide feeding in preterm infants, but there are few data demonstrating their usefulness. Similarly, techniques are now available to investigate gastrointestinal (GI) function noninvasively and safely, but their ability to predict attainment of full gavage feedings and/or feeding volume in preterm infants is unclear., Objective: We sought to determine prospectively the potential relationships of attainment of full gavage feedings and feeding volume with clinical measures and noninvasive GI tests., Methods: Fifty preterm infants were followed prospectively. Daily tally was taken of gavage feeding intake, gastric residual volumes (GRVs; milliliters per day, number of GRVs >50% of the previous feeding volume, and number of GRVs >2 ml/kg), and abdominal distention. Infants underwent repeated measurement of lactase activity, GI permeability, fecal calprotectin concentration, and gastric emptying., Results: The number of GRVs >2 ml/kg tended to decrease with postnatal age (p = 0.06). Lactase activity and feeding volume in milliliters per kilogram per day prior to achieving full feedings were correlated (p = 0.007, β = 0.164). There was no correlation between feeding outcomes and GRV (ml/day), GRV >50%, GRV >2 ml/kg, small bowel, colonic, or whole bowel permeability, fecal calprotectin concentration, gastric emptying, or abdominal distention., Conclusions: GRV is unreliable in predicting attainment of full gavage feeding. Lactase activity is related to feeding volume. However, other noninvasive GI tests utilized were not predictive. These data cast doubt upon the utility of GRV in guiding feeding therapy. Randomized trials of different GRV management protocols are needed., (Copyright © 2010 S. Karger AG, Basel.)

Therapeutic regulation of globin genes is a primary goal of translational research aimed toward hemoglobinopathies. Signal transduction was used to identify chromatin modifications and transcription factor expression patterns that are associated with globin gene regulation. Histone modification and transcriptome profiling were performed using adult primary CD34(+) cells cultured with cytokine combinations that produced low versus high levels of gamma-globin mRNA and fetal hemoglobin (HbF). Embryonic, fetal, and adult globin transcript and protein expression patterns were determined for comparison. Chromatin immunoprecipitation assays revealed RNA polymerase II occupancy and histone tail modifications consistent with transcriptional activation only in the high-HbF culture condition. Transcriptome profiling studies demonstrated reproducible changes in expression of nuclear transcription factors associated with high HbF. Among the 13 genes that demonstrated differential transcript levels, 8 demonstrated nuclear protein expression levels that were significantly changed by cytokine signal transduction. Five of the 8 genes are recognized regulators of erythropoiesis or globin genes (MAFF, ID2, HHEX, SOX6, and EGR1). Thus, cytokine-mediated signal transduction in adult erythroid cells causes significant changes in the pattern of globin gene and protein expression that are associated with distinct histone modifications as well as nuclear reprogramming of erythroid transcription factors.

Objectives: Tropical enteropathy is characterized by an increased urinary lactulose-to-mannitol (L:M) ratio on a site-specific sugar absorption test and is associated with increased intestinal permeability and decreased nutrient absorptive capacity. The etiology of tropical enteropathy is postulated to be intestinal bacterial overgrowth. This study tested the hypothesis that treatment with a nonabsorbable, broad-spectrum antibiotic, rifaximin, reduces the L:M ratio in rural Malawian children, among whom tropical enteropathy is common., Methods: All children aged 3-5 years from one village were enrolled in a randomized, double-blind, placebo-controlled trial of treatment with rifaximin for 7 days. The L:M ratio was measured before and after treatment, and the change in the L:M ratio was the primary outcome. Secondary outcomes were changes in the urinary sucrose-to-lactulose (SUC:L) and sucralose-to-lactulose (SCL:L) ratios, as well as changes in the fractions of each test sugar recovered in the urine., Results: A total of 144 children participated in this study, of whom 76% had an elevated L:M ratio on enrollment (L:M > or = 0.10). Children who received rifaximin did not show an improvement in their L:M ratio compared with those who received placebo (-0.01+/-0.12 vs. 0.02+/-0.16, respectively, P=0.51, mean+/-s.d.), nor were there significant differences between the two groups in excretion of lactulose, mannitol, sucralose, or sucrose, or in the SUC:L and SCL:L ratios., Conclusions: Rifaximin had no effect on the tropical enteropathy of 3-5-year-old Malawian children, suggesting that small-bowel bacterial overgrowth is not an important etiological factor in this condition.

Objectives: To determine gastrointestinal (GI) permeability and fecal calprotectin concentration in children 7 to 10 years of age with functional abdominal pain and irritable bowel syndrome (FAP/IBS) versus control subjects and ascertain potential relationships with pain symptoms and stooling., Study Design: GI permeability and fecal calprotectin concentration were measured. Children kept a 2-week diary of pain episodes and stooling pattern., Results: Proximal GI permeability was greater in the FAP/IBS group (n = 93) compared with control subjects (n = 52) (0.59 +/- 0.50 vs 0.36 +/- 0.26, respectively; mean +/- SD; P < .001) as was colonic permeability (1.01 +/- 0.67 vs 0.81 +/- 0.43, respectively; P < .05). Gastric and small intestinal permeability were similar. Fecal calprotectin concentration was greater in children with FAP/IBS compared with control children (65.5 +/- 75.4 microg/g stool vs 43.2 +/- 39.4, respectively; P < .01). Fecal calprotectin concentration correlated with pain interference with activities (P = .01, r(2) = 0.36). There was no correlation between GI permeability and pain related symptoms. Neither permeability nor fecal calprotectin correlated with stool form., Conclusions: Children with FAP/IBS have evidence of increased GI permeability and low-grade GI inflammation, with the latter relating to the degree to which pain interferes with activities.

The Sebia Capillarys (capillary zone electrophoresis [CE]) and the Primus Resolution high-pressure liquid chromatography (HPLC) were used to prospectively evaluate 297 samples for hemoglobinopathies. Hemoglobin (Hb) A levels were similar on both techniques (mean, 96.2% and SD, 5.7% by CE; mean, 96.8% and SD, 5.5% by HPLC), but HbA2 levels were higher by CE (mean, 2.8%; SD, 0.8%) than by HPLC (mean, 2.3%; SD, 0.8%). HbS had higher values by CE (mean, 40.6%; SD, 18.9%) than by HPLC (mean, 38.4%; SD, 18.9%). In cases with Hg S, HbA2 levels were greater by HPLC (mean, 4.0%; SD, 1.0%) than by CE (mean, 3.1%; SD, 0.8%). HbA2 was occasionally not separated sufficiently from HbC for measurement by CE, but did separate from HbE by CE. Both methods identified HbS, HbC, HbE, HbS, and HbC together, HbA2&prime, HbD-Los Angeles, HbF variant, HbG-Philadelphia, HbS-G Philadelphia, and Hb Lepore.

Lactobacillus reuteri is a commensal-derived anaerobic probiotic that resides in the human gastrointestinal tract. L. reuteri converts glycerol into a potent broad-spectrum antimicrobial compound, reuterin, which inhibits the growth of gram-positive and gram-negative bacteria. In this study, we compared four human-derived L. reuteri isolates (ATCC 55730, ATCC PTA 6475, ATCC PTA 4659 and ATCC PTA 5289) in their ability to produce reuterin and to inhibit the growth of different enteric pathogens in vitro. Reuterin was produced by each of the four L. reuteri strains and assessed for biological activity. The minimum inhibitory concentration (MIC) of reuterin derived from each strain was determined for the following enteric pathogens: enterohemorrhagic Escherichia coli, enterotoxigenic E. coli, Salmonella enterica, Shigella sonnei and Vibrio cholerae. We also analyzed the relative abilities of L. reuteri to inhibit enteric pathogens in a pathogen overlay assay. The magnitude of reuterin production did not directly correlate with the relative ability of L. reuteri to suppress the proliferation of enteric pathogens. Additional antimicrobial factors may be produced by L. reuteri, and multiple factors may act synergistically with reuterin to inhibit enteric pathogens.

Purpose: Valproic acid (VPA), a widely used antiepileptic, also inhibits histone deacetylase (HDAC), and is undergoing evaluation as an anti-cancer agent. We studied the pharmacokinetics of VPA in the plasma and cerebrospinal fluid (CSF) in a non-human primate model that is highly predictive of human CSF penetration to determine if levels of VPA required to inhibit HDAC in in vivo models can be attained., Methods: Oral VPA, 75 mg/kg, was administered to four non-human primates. Serial samples of blood (n = 4) and CSF (n = 3) were obtained for pharmacokinetic studies of total and free VPA. Plasma and CSF VPA concentrations were measured using the commercially available Abbott AxSYM VPA assay reagent system (Abbott Laboratories, Abbott Park, IL, USA). The resultant plasma and CSF data were evaluated using pharmacokinetic modeling methods., Results: At a dose of 75 mg/kg, the maximum plasma concentration of VPA was 130.1 +/- 70.6 microg/ml (mean +/- standard deviation) for total drug and 53.3 +/- 44.4 microg/ml for free drug. The mean plasma area under the curve (AUC) for total drug was 680 +/- 233 microg/ml h and for free drug 146 +/- 89 microg/ml hr. The maximum CSF concentration occurred 2-3 h after administration and was 28.2 +/- 18.6 microg/ml. The CSF AUC for VPA was 108 +/- 52 microg/ml h. The CSF penetration of VPA was 12.9 +/- 5.1% for total drug and 57.0 +/- 8.7% for free drug. Disappearance from the plasma followed non-linear kinetics with a V (max) of 321.2 +/- 65.6 microg/kg/min and a K (m) of 17.2 +/- 13.7 mg/l., Conclusion: Valproic acid deserves further study for the treatment of CNS tumors given its high CSF penetration after oral dosing coupled with the anti-tumor activity observed in preclinical studies.

Hemoglobin Monroe (beta globin G->C, codon 30) is a missense mutation. We could not detect either the mutant peptide or transcript in reticulocyte-enriched preparation and in expanded erythroid progenitor cells. By quantitative gene expression assay beta globin mRNA was found to be reduced by more than 70% in all heterozygous subjects with different haplotypes. We conclude that this mutation also interferes with expression of wild type allele.

Two polycythemic subjects from a family with multiple polycythemic subjects were evaluated. Estimation of oxygen affinity of Hb from venous blood gas parameters (P50) revealed low P50 suggesting a high affinity Hb variant. Further work up, which included beta globin gene sequencing, revealed a novel mutation changing a codon to the previously reported high affinity Hb - Hb Johnstown (beta 109 Val->Leu). Polycythemic subjects with high affinity Hb variant are asymptomatic with normal life expectancy. Their differentiation from polycythemia vera (PV) is crucial to avoid therapy which is otherwise reserved for PV patients. We provide an electronic version (in Microsoft excel program) of a previously reported mathematical formula for rapid calculation of P50 from venous blood gases. Estimation of P50 is an essential initial step in the evaluation of a subject with personal and family history of polycythemia.

Objectives: It is known that levels of brain natriuretic peptide predict outcomes of treatment for adults with decompensated heart failure. We hypothesized that it could predict outcomes in children with this condition., Methods: We divided retrospectively 82 patients with serial measurements of brain natriuretic peptide into 3 groups: those who survived and did not need readmission within less than 60 days; those who survived but needed readmission within less than 60 days; and those who died in hospital or within less than 60 days. Initial and final levels of the peptide correlated with adverse outcomes., Results: The percent change in level of the peptide was minus 78 percent, minus 38 percent, and 138 percent in the readmission-free group, the readmitted, and nonsurviving groups, respectively. Final levels were significantly lower in the readmission-free group than in the readmitted and nonsurviving groups (p equals 0.013 and p is less than 0.00001, respectively) and in the readmitted group than in the nonsurvivors (p equals 0.013). On univariate analysis, the final level, the change in level, and the percentage change in level significantly predicted outcomes (p equals 0.0002, 0.0072 and 0.0005, respectively). On multivariate analysis, only the final level of the peptide significantly predicted outcomes (p equals 0.01)., Conclusions: A final level of brain natriuretic peptide of greater than or equal to 760 picograms per millilitre strongly predicted an adverse outcome. Patients with higher final levels may be at higher risk of death and readmission, suggesting that this variable effectively predicts the response to treatment and prognosis in children with heart failure.

Background: Pulse oximetry is commonly used to monitor oxygenation in neonates, but cannot detect variations in hemoglobin. Venous and arterial oxygen saturations are rarely monitored. Few data are available to validate measurements of oxygen saturation in neonates (venous, arterial, or pulse oximetric). Purpose To validate oxygen saturation displayed on clinical monitors against analyses (with correction for fetal hemoglobin) of blood samples from neonates and to present the oxyhemoglobin dissociation curve for neonates., Method: Seventy-eight neonates, 25 to 38 weeks' gestational age, had 660 arterial and 111 venous blood samples collected for analysis., Results: The mean difference between oxygen saturation and oxyhemoglobin level was 3% (SD 1.0) in arterial blood and 3% (SD 1.1) in venous blood. The mean difference between arterial oxygen saturation displayed on the monitor and oxyhemoglobin in arterial blood samples was 2% (SD 2.0); between venous oxygen saturation displayed on the monitor and oxyhemoglobin in venous blood samples it was 3% (SD 2.1) and between oxygen saturation as determined by pulse oximetry and oxyhemoglobin in arterial blood samples it was 2.5% (SD 3.1). At a Pao(2) of 50 to 75 mm Hg on the oxyhemoglobin dissociation curve, oxyhemoglobin in arterial blood samples was from 92% to 95%; oxygen saturation was from 95% to 98% in arterial blood samples, from 94% to 97% on the monitor, and from 95% to 97% according to pulse oximetry., Conclusions: The safety limits for pulse oximeters are higher and narrower in neonates (95%-97%) than in adults, and clinical guidelines for neonates may require modification.

Background: The purposes of this study were to examine the accuracy of fetal hemoglobin (HbF) as quickly measured by the hemoximeter, verified by the high-performance liquid chromatography method, and to examine related oxygen saturation (SO(2)) measurements in neonates., Methods: Thirty-nine neonates with gestational ages ranging from 25 to 38 weeks were investigated (n=280 blood samples). Twenty younger premature neonates had blood transfusions (n=188 blood samples, 72 before and 116 after transfusions), and 19 older neonates did not., Results: The bias of the hemoximeter was 23% (+/-9.1) against the HPLC; 25% (+/-7.9) before, and 19% (+/-8.6) after blood transfusions (all P<0.001), for HbF measurements. A regression line (HbFt by the HPLC=8.46+0.7 x HbF by the hemoximeter) has been provided for the prediction. Oxyhemoglobin dissociation curves with the status of (before and after) blood transfusions were presented. In relation to oxygen tension values of 50-75 mm Hg, in addition to the right-shifted oxyhemoglobin dissociation curves, pulse oximeter ranged from 95 to 98% before the transfusions, but decreased to 94 to 96% after the blood transfusions., Conclusions: Accurate HbF and related oxygen saturation measurements need to be determined, especially for premature neonates, to minimize the risk of oxygen toxicity.

Purpose: To develop novel orthotopic xenograft models of medulloblastoma in severe combined immunodeficient mice and to evaluate the in vivo antitumor efficacy of valproic acid., Experimental Design: Orthotopic xenografts were developed by injecting 10(3) to 10(5) tumor cells from four medulloblastoma cell lines (D283-MED, DAOY, MHH-MED-1, and MEB-MED-8A) into the right cerebellum of severe combined immunodeficient mice. Animals were then examined for reproducibility of tumorigenicity, cell number-survival time relationship, and histopathologic features. Tumor growth was monitored in vivo by serially sectioning the xenograft brains at 2, 4, 6, and 8 weeks postinjection. Valproic acid treatment, administered at 600 microg/h for 2 weeks via s.c. osmotic minipumps, was initiated 2 weeks after injection of 10(5) medulloblastoma cells, and treated and untreated animals were monitored for differences in survival. Changes in histone acetylation, proliferation, apoptosis, differentiation, and angiogenesis in xenografts were also evaluated., Results: Tumorigenicity was maintained at 100% in D283-MED, DAOY, and MHH-MED-1 cell lines. These cerebellar xenografts displayed histologic features and immunohistochemical profiles (microtubule-associated protein 2, glial fibrillary acidic protein, and vimentin) similar to human medulloblastomas. Animal survival time was inversely correlated with injected tumor cell number. Treatment with valproic acid prolonged survival time in two (D283-MED and MHH-MED-1) of the three models and was associated with induction of histone hyperacetylation, inhibition of proliferation and angiogenesis, and enhancement of apoptosis and differentiation., Conclusion: We have developed intracerebellar orthotopic models that closely recapitulated the biological features of human medulloblastomas and characterized their in vivo growth characteristics. Valproic acid treatment of these xenografts showed potent in vivo anti-medulloblastoma activity. These xenograft models should facilitate the understanding of medulloblastoma pathogenesis and future preclinical evaluation of new therapies against medulloblastoma.

The purposes of this study were to examine the accuracy of fetal hemoglobin (Hb F, alpha2gamma2) as quickly measured by a hemoximeter but verified by high performance liquid chromatography [HPLC, including Hb F total (Hb Ft), acetylated Hb F (Hb F1), and non acetylated Hb F ( Hb F*)], and to predict the Hb F levels for different gestational weeks of neonates. Thirty-nine neonates of predominantly Hispanic and African American ethnicity, with gestational ages ranging from 25 to 38 weeks, were investigated. Analyses were performed on 163 blood samples that were pure neonates' blood before the transfusion of any adult blood. Two neonates had increased Hb C [beta6(A3)Glu-->Lys, GAC-->AAG] levels (1.67-2.79%) and one neonate whose mother drank alcohol during pregnancy, had elevated Hb A2 levels (0.12-0.14%). After excluding these data points, the mean Hb F were overestimated by hemoximeter, 118.4 +/- 8.77% vs. 92.6 +/- 2.77% by HPLC (mean difference: 25.8 +/- 7.71%, p = <0.001). Mean Hb F1 was 10.5 +/- 2.28%. Hb F levels decreased as gestational age increased (p <0.001 for Hb Ft and Hb F*; p = <0.05 for Hb F1). A multivariate regression model for Hb F prediction was established with the best R2. The gestational age and post birth hours in the prediction of Hb Ft was included when Hb F could be determined at the clinical settings. Future studies may be needed to account for Hb F1 when measuring Hb F levels to assess oxygenation status in (pre term) neonates.

Background: Tropical enteropathy is an asymptomatic villous atrophy of the small bowel that is prevalent in the developing world and is associated with altered intestinal function and integrity. The histology of tropical enteropathy resembles that seen in small-bowel bacterial overgrowth., Objective: This study tested the hypothesis that treatment of 3-5-y-old Malawian children with the probiotic Lactobacillus GG would improve their intestinal function and integrity., Design: Clinically healthy children (n = 164) were enrolled in a placebo-controlled, randomized, double-blind trial. Intestinal function and integrity were measured by using the site-specific sugar-absorption test before and after 30 d of treatment with Lactobacillus GG or placebo. The primary outcomes were the ratios of urinary lactulose to mannitol (L:M) and of urinary sucrose to lactulose (S:L) excretion., Results: Of the 161 children who completed the study, 119 (73%) had tropical enteropathy on enrollment (L:M > 0.10). Children receiving Lactobacillus GG did not differ significantly from the placebo group in the excretion (in % of dose administered) of mannitol (mean +/- SD: 8.9 +/- 4.4 and 8.9 +/- 3.9, respectively), lactulose (0.31 +/- 0.20 and 0.33 +/- 0.23, respectively), or sucrose (0.078 +/- 0.058 and 0.082 +/- 0.075, respectively). L:M and S:L also did not differ significantly between the Lactobacillus and placebo groups (0.19 +/- 0.13 and 0.20 +/- 0.12, respectively, for L:M; 0.58 +/- 0.46 and 0.65 +/- 0.57, respectively, for S:L)., Conclusion: Administration of Lactobacillus GG for 30 d had no effect on the intestinal integrity of 3-5-y-old Malawian children.

Many pediatric anesthesiologists divide acetaminophen suppositories to achieve an approximate dose. In this three-part study we first surveyed pediatric anesthesiologists regarding their attitudes and frequency of this clinical practice. Second, acetaminophen suppositories were divided for analysis of acetaminophen content. Finally, the accuracy of pediatric anesthesiologists in dividing suppositories was assessed. The survey indicated 50% of anesthesiologists believed acetaminophen was nonuniform and 62% believed the alteration of suppositories was inaccurate. The laboratory investigation revealed uniform distribution of acetaminophen but poor accuracy in achieving the target dose. The findings suggest using only intact suppositories for improved accuracy.

The murine model of hereditary tyrosinemia type 1 (HT1) was used to analyze the relationship between chronic liver disease and programmed cell death in vivo. In healthy fumarylacetoacetate hydrolase deficient mice (Fah(-/-)), protected from liver injury by the drug 2-(2- nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), the tyrosine metabolite homogentisic acid (HGA) caused rapid hepatocyte death. In contrast, all mice survived the same otherwise lethal dose of HGA if they had preexisting liver damage induced by NTBC withdrawal. Similarly, Fah(-/-) animals with liver injury were also resistant to apoptosis induced by the Fas ligand Jo-2 and to necrosis-like cell death induced by acetaminophen (APAP). Molecular studies revealed a marked up-regulation of the antiapoptotic heat shock proteins (Hsp) 27, 32, and 70 and of c-Jun in hepatocytes of stressed mice. In addition, the p38 and Jun N-terminal kinase (JNK) stress-activated kinase pathways were markedly impaired in the cell-death resistant liver. In conclusion, these results provide evidence that chronic liver disease can paradoxically result in cell death resistance in vivo. Stress-induced failure of cell death programs may lead to an accumulation of damaged cells and therefore enhance the risk for cancer as observed in HT1 and other chronic liver diseases.

Sepsis is the most common morbidity in preterm infants, who often receive total parenteral nutrition (TPN). We hypothesized that gut barrier function is compromised in TPN-fed compared with enterally fed newborn piglets (ENT pigs). Colostrum-deprived newborn pigs were implanted with jugular venous and bladder catheters under general anesthesia. Pigs were either administered TPN (n = 15) or fed formula (ENT pigs, n = 15). After 6 days, pigs were gavaged a solution of mannitol, lactulose, and polyethylene glycol 4000 (PEG 4000) and urine was collected for 24 h. At 7 days, small bowel samples were assayed for myeloperoxidase activity, morphometry, and tight junction protein abundance. Intestinal contents and peripheral organ sites were cultured for bacteria. Urinary recovery (%dose) of mannitol (53 vs. 68) was lower, whereas that of lactulose (2.93 vs. 0.18) and PEG 4000 (12.78 vs. 0.96) were higher in TPN vs. ENT pigs, respectively (P < 0.05). Incidence of translocation was similar in TPN and ENT pigs. Myeloperoxidase activity was increased in TPN vs. ENT pigs in the jejunum (P < 0.001) and was weakly correlated with lactulose (R2 = 0.32) and PEG 4000 (R2 = 0.38) recovery. Goblet cell counts did not change, but intraepithelial lymphocyte numbers decreased with TPN. Only claudin-1 protein abundance was increased in the TPN group. We conclude that TPN is associated with impairment of neonatal gut barrier function as measured by permeability but not translocation.

Background: Anti-glomerular basement membrane (GBM) glomerulonephritis is among the earliest recognized human autoimmune diseases. However, the etiology of anti-GBM glomerulonephritis remains unclear. We have previously shown that CD4+ T cells, specific to alpha3 NC1 of type IV collagen (Col4alpha3NC1), were able to induce anti-GBM glomerulonephritis in Wistar-Kyoto (WKY) rats. In the present study, we continued to map the nephritogenic T cell epitope in Col4alpha3NC1., Methods: Synthetic peptides, which covered Col4alpha3NC1, were used as immunogens to induce glomerulonephritis in WKY rats. T-cell and B-cell responses to the peptides in the animals were analyzed., Results: One potent nephritogenic T-cell epitope, pCol(28-40) (SQTTANPSCPEGT), was identified. A single immunization with pCol(28-40) induced extremely severe glomerulonephritis in all 23 rats. Renal pathology revealed nearly 100% of glomeruli with crescentic lesions or tuft necrosis in 21 animals. pCol(28-40) elicited a T-cell response to the peptide; T cells isolated from rats immunized with recombinant Col4alpha3NC1 reacted with pCol(28-40). pCol(28-40) elicited a peptide specific antibody response, which did not react with polypeptide Col4alpha3NC1 or native GBM. An 11-mer peptide, pCol(a30-40) (Ac-TTANPSCPEGT), was further mapped to be the core of the T-cell epitope in pCol(28-40). As expected, immunization with pCol(a30-40) induced severe glomerulonephritis in 10 out of 19 rats., Conclusion: Our study not only demonstrated that a single T-cell epitope of Col4alpha3NC1 is sufficient to induce severe glomerulonephritis, but also provides a unique model for studying T-cell-mediated mechanisms in anti-GBM glomerulonephritis pathogenesis.

Susceptibility of activated T cells to apoptosis must be tightly regulated to ensure sufficient T cell progeny for an effective response, while allowing a rapid depletion of them at the end of the immune response. We show here that a previously isolated, NF-kappa B/rel target gene IEX-1 (Immediate Early response gene X-1) is highly expressed in T cells at early stages of activation, but declines with a prolonged period of activation time, coincident with an increased susceptibility of T cells to apoptosis during the late phases of an immune response. Transgenic expression of IEX-1 specifically in lymphocytes impaired apoptosis in activated T cells, extended a duration of an effector-phase of a specific immune response, and increased the accumulation of effector/memory-like T cells and the susceptibility to a lupus-like autoimmune disease. Our study demonstrated an antiapoptotic effect of IEX-1 on T cell apoptosis triggered by ligation of Fas and T cell receptor (TCR)/CD3 complex. The ability of extending life expectancy of T effectors, in line with a decrease in its expression following prolonged T cell activation, suggests a key role for IEX-1 in regulating T cell homeostasis during immune responses.

Background: A mycobacterial infection may be the cause of Crohn's disease in some patients. Measurement of intestinal permeability may identify Crohn's disease patients with a high likelihood of relapse and may quantify the severity of intestinal injury., Aim: To assess the effect of 3 months of clarithromycin and ethambutol on the disease activity and intestinal permeability in patients with Crohn's disease at high risk of relapse., Methods: Patients with Crohn's disease, with a lactulose-mannitol permeability test above 0.03, were randomly assigned to receive either clarithromycin, 500 mg twice daily, and ethambutol, 15 mg/kg daily, or identically appearing placebo for 3 months in addition to their regular therapy. The Harvey-Bradshaw index and the lactulose-mannitol test were assessed in a blind fashion every 3 months for 12 months., Results: Thirty-one patients were randomized to receive either drugs (n=15) or placebo (n=16). The groups were similar in age, sex, duration of disease, location of disease, past complications and disease severity. Specifically, there was no difference between the drug or placebo groups in the mean Harvey-Bradshaw index (4.8 vs. 4.4), number with active disease (33% vs. 44%) and mean lactulose-mannitol test (0.06 vs. 0.10). During the 12-month follow-up period, there were no consistent, statistically significant differences in the mean Harvey-Bradshaw index or lactulose-mannitol test between treatment and placebo groups. Individual patients showed either improvement or worsening of these indices, but these were not related to study medication. Specifically, no 'cures' were noted with anti-mycobacterial treatment., Conclusions: Three months of treatment with clarithromycin and ethambutol does not benefit Crohn's disease patients who are receiving standard medical therapy.

We have proposed that the nephrotoxicity of cisplatin, a widely used chemotherapy drug, is the result of the binding of cisplatin to glutathione and the subsequent metabolism of the cisplatin-glutathione complex via a gamma-glutamyl transpeptidase (GGT)-dependent pathway in the proximal tubules. To test the hypothesis that GGT activity is essential for the nephrotoxicity of cisplatin, the effects of cisplatin were examined in wild-type and GGT-deficient mice. Mice were treated with 15 mg cisplatin/kg. Five days after treatment, renal histopathology, blood urea nitrogen levels, serum creatinine, platinum excretion, and platinum accumulation in the kidney were examined. Half the mice were supplemented with N-acetylcysteine, which has been shown to correct low levels of tissue glutathione in GGT-deficient mice. The data show that cisplatin was nephrotoxic in wild-type mice but not in GGT-deficient mice. The wild-type mice, with and without N-acetylcysteine supplementation, had significantly elevated levels of blood urea nitrogen, serum creatinine, and renal tubular necrosis. There was no evidence of nephrotoxicity in the GGT-deficient mice regardless of N-acetyl cysteine supplementation. No differences in platinum excretion were seen comparing wild-type and GGT-deficient mice, nor was there any significant difference in renal platinum accumulation. These data suggest that renal cisplatin toxicity is dependent on GGT activity, and is not correlated with uptake. The results support our hypothesis that the nephrotoxicity of cisplatin is the result of the metabolism of the drug through a GGT-dependent pathway.

Background: Alkaline cellulose acetate and acidic citrate agar electrophoreses are the most widely utilized methods for hemoglobin analysis. However, due to their limited resolution, incorrect or unresolved diagnosis of common hemoglobinopathies are sometimes encountered., Methods: Isoelectric focusing provides excellent resolution but is labor intensive and lacks accurate quantitation. High-performance liquid chromatographic methods have been developed for either screening or confirmation of hemoglobinopathies with relatively high sensitivity or specificity. Through the years, we have developed, refined and optimized an HPLC procedure using a porous silica coated with polyaspartic acid to improve the elution time of hemoglobin analysis while maintaining the high sensitivity and resolution necessary for both screening and confirmatory purposes., Results: The method is capable of separating more than 45 commonly encountered hemoglobin variants within 12 min. These include Barts, H, A1C, Raleigh, Hope, I, F, Camden, N-Baltimore, I-High Wycombe, I-Paris, J-Baltimore, N-Seattle, Grade, Fannin-Lubbock, Malmo, South Florida, A, Chicago, G-Georgia, Lepore-Baltimore, P-Galveston, G-Coushatta, Lepore-Boston, E, Zurich, Osu Christiansborg, A2, G-Philadelphia, Korle Bu, Russ, E-Saskatoon, Richmond, D-Punjab, Deer Lodge, Koln, Montgomery, S, Q-Thailand, G-San Jose, A2', Hasharon, Q-India, Tampa, Constant Spring, SG-hybrid, C-Harlem, O-Arab, British Columbia, and C. The method provides not only the identification of the aforementioned hemoglobin and variants but also an accurate quantitation of their concentrations, particularly Hb F and A2, which are useful for the diagnosis of HPFH and beta-thalassemia, respectively., Conclusions: The simplicity of the sample preparation, superior resolution of the method, and accurate quantitation of hemoglobin concentration, combined with complete automation, make this an ideal methodology for the routine diagnosis of hemoglobin disorders in a clinical laboratory.

A combination of chromatographic and mass spectrometric techniques was used to evaluate the extent and distribution of glycation within the glycated hemoglobin (GHb) molecule. Studies on quantification of hemoglobin (Hb) glycation by electrospray ionization mass spectrometry (ES-MS) of intact globins employed specimens from 10 diabetic individuals and five normal controls. Detailed structural analysis of the phenylboronate affinity chromatography/ion-exchange (IE) HPLC-separated sub-populations of GHb was performed on a specimen carrying 13.7% GHb. An efficient protocol for mapping glycation sites within alpha and beta globins was developed, e.g., Glu-C/Asp-N proteolytic digestion followed by LC-ES-MS. Relative site occupancy within discrete components of GHb was evaluated. A correlation between the degree of glycation measured at Hb level (by affinity chromatography) and at globin level (measured by ES-MS) was carried out. The above studies led us to conclude that during the process of phenylboronate chromatography GHb dimers, rather than tetramers, are bound to the affinity resin so a fraction of glycated dimers rather than tetramers is measured. This finding implies that a process of glycation affects a much higher number of native Hb tetramers than was previously contemplated. No glycation sites appear to be missed by phenylboronate affinity chromatography. We have found no evidence of the presence of multiple glycations within a single globin chain. While glycation of both globins within a dimer cannot be excluded, it is unlikely to be a significant phenomenon. According to ES-MS data, an equivalent of about one globin per alphabeta dimer of the affinity chromatography-isolated GHb carried glycation.

Drosophila dachshund is necessary and sufficient for compound eye development and is required for normal leg and brain development. A mouse homologue of dachshund, Dach1, is expressed in the developing retina and limbs, suggesting functional conservation of this gene. We have generated a loss-of-function mutation in Dach1 that results in the abrogation of the wild-type RNA and protein expression pattern in embryos. Homozygous mutants survive to birth but exhibit postnatal lethality associated with a failure to suckle, cyanosis, and respiratory distress. The heart, lungs, kidneys, liver, and skeleton were examined to identify factors involved in postnatal lethality, but these organs appeared to be normal. In addition, blood chemistry tests failed to reveal differences that might explain the lethal phenotype. Gross examination and histological analyses of newborn eyes, limbs, and brains revealed no detectable abnormalities. Since Dach1 mutants die shortly after birth, it remains possible that Dach1 is required for postnatal development of these structures. Alternatively, an additional Dach homologue may functionally compensate for Dach1 loss of function.

We used mice with a targeted disruption in g-glutamyl transpeptidase (GGT-deficient mice) to study the role of glutathione (GSH) in protection against oxygen-induced lung injury. These mice had reduced levels of lung GSH and restricted ability to synthesize GSH because of low levels of cysteine. When GGT-deficient mice were exposed to 80% oxygen, they developed diffuse pulmonary injury and died within eight days. Ten of 12 wild-type mice were alive after 18 days. Administration of N-acetylcysteine (NAC) to GGT-deficient mice corrected GSH values and prevented the development of severe pulmonary injury and death. Oxygen exposure induced an increase in lung GSH levels in both wild-type and GGT-deficient mice, but induced levels in the mutant mice were <50% of those in wild-type mice. Cysteine levels were approximately 50-fold lower than GSH levels the lungs of both wild-type and GGT-deficient mice. Levels of lung RNA coding for the heavy subunit of g-glutamyl cysteine synthetase rose three- to fourfold after oxygen exposure in both wild-type and GGT-deficient mice. In contrast, oxygen exposure failed to provoke increases in glutathione synthetase, glutathione peroxidase, glutaredoxin, or thioredoxin.

The present study was undertaken to analyse the relationship of lens glutathione (GSH) and light to cataract development in mice deficient in gamma-glutamyl transpeptidase (GGT). These mice have reduced levels of cysteine and GSH in the eye and develop cataracts. GGT-deficient mice raised under normal vivarium conditions, showed no cataractous changes at birth, but by 1 week they had developed nuclear opacities. By 3 weeks more severe cataracts develop, and lens GSH levels are approximately 6-7% of wild type levels. By 6-11 weeks cataracts show nuclear and cortical involvement, liquefaction and calcification. Single cell DNA electrophoresis (comet assay) demonstrated mild DNA damage in the lens epithelium. GGT-deficient mice raised in the dark beginning the day after conception all developed cataracts, but these were less severe than those in GGT-deficient mice raised with normal vivarium lighting. Administration of N -acetyl cysteine (NAC) raises lens GSH and almost completely prevents cataract development. Our data indicate that cataract development in GGT-deficient mice is multifactorial and results from exogenous damage (exposure to light), reduced lens GSH levels, and nutritional effects secondary to low cysteine levels., (Copyright 2000 Academic Press.)

Recent studies have shown that expression levels of the multidrug resistance gene MDR1, which encodes the drug transporter P-glycoprotein, correlate with prognostic outcomes of certain tumor types. These findings suggest that expression of MDR1 may affect tumor behaviors. To address this issue further, we investigated the expression of mdr1a, a human MDR1 homolog, on the development of hepatocellular carcinoma in a transgenic mouse model carrying the liver-targeted expression of human hepatitis-B virus (HBV) surface antigen. The pathogenetic program was compared in HBV mice carrying either mdr1a(+/+) or mdr1a(-/-). We found that the expressions of proliferative activity markers, Ki67 nuclear antigen, and proliferating cell nuclear antigen were elevated in mdr1a(-/-) mice younger than 10 wk in comparison with those in the same age group of wild-type animals. Replication in the hepatic population as determined by bromodeoxyuridine incorporation tended to support observation that mdr1a(-/-) mice exhibited elevated labeling indices in this age group. Moreover, histologic staining and flow-cytometric analysis showed that the mdr1a(-/-) animals exhibited a higher cell population with polyploidy than did the mdr1a(+/+) counterparts of the same age. However, no significant differences in the expression of the liver-injury markers serum alanine transaminase and aspartate transaminase were observed. Although our results showed that absence of mdr1a expression is correlated with modest enhanced proliferative characteristics in the livers at stage before the development of hepatocellular carcinoma, the overall life spans between these two strains of mice were not significantly different. The implication of these findings to the role of P-glycoprotein in tumor development and cancer chemotherapy is discussed.

We used mice deficient in gamma-glutamyl transpeptidase (GGT) to analyze the effects of GGT deficiency and altered thiol levels on gene expression in liver. GGT-deficient mice have markedly reduced levels of glutathione (GSH), cysteine, methionine, and cysteinylglycine in liver. Steady-state RNA levels of the catalytic subunit of gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme in GSH synthesis, are elevated 4-fold in these mice, while those for glutathione synthetase (GSH syn) are elevated 2-fold. RNA levels of cystathionase (cystathionine gamma-lyase), a key enzyme in the synthesis of cysteine from methionine, are elevated approximately 3.5-fold. In contrast, levels of RNA coding for multidrug resistance protein 2 (MRP2), which transports GSH into bile, are half wild-type values. We found no change in RNA levels of enzymes related to oxidative injury (CuZn and Mn superoxide dismutases [SOD], catalase, and glutathione peroxidase). Similarly, RNA levels of glutathione reductase and ribonucleotide reductase were unchanged. Furthermore, in contrast to previous in vitro results, methyl methanesulfonate did not induce stress-activated signal transduction as measured by c-jun phosphorylation in livers of GGT-deficient mice, despite further depletion of GSH by buthionine sulfoximine. Our findings indicate that GGT deficiency itself and/or altered thiol levels regulate expression of genes involved in GSH metabolism, but have no effect on the expression of other antioxidant genes.

Sickle cell disease is a hereditary disorder characterized by erythrocyte deformity due to hemoglobin polymerization. We assessed in vivo the potential curative threshold of fetal hemoglobin in the SAD transgenic mouse model of sickle cell disease using mating with mice expressing the human fetal Agamma-globin gene. With increasing levels of HbF, AgammaSAD mice showed considerable improvement in all hematologic parameters, morphopathologic features and life span/survival. We established the direct therapeutic effect of fetal hemoglobin on sickle cell disease and demonstrated correction by increasing fetal hemoglobin to about 9-16% in this mouse model. This in vivo study emphasizes the potential of the SAD mouse models for quantitative analysis of gene therapy approaches.

Gastrointestinal bleeding and increased intestinal permeability have been observed in marathon runners. We sought to determine if L-arginine would be useful for prevention of these complications. Twenty-three runners were randomized to receive L-arginine (A) or glycine (placebo) (G), 10 grams 3 times daily for 14 days prior to the 1997 Houston-Methodist Marathon. Serum, stool hemoccults and lactulose:mannitol permeabilities were obtained at baseline, immediately after completion of the marathon and approximately 48 hours later. Runners rated their symptoms of nausea and vomiting, belching and indigestion, abdominal pain and bloating, diarrhea, and extremity pain on a 1-5 scale of increasing severity. The L:M was unchanged in either group during the three collections. Occult bleeding occurred in 8%/20% in A and G groups, respectively, p = NS) immediately post-marathon. No runners had occult bleeding 48 hours post-race. Gastrointestinal symptom scores were minimal to nonexistent. Extremity pain scores were similar for groups A and G (2.1+/-1.4 and 2.8+/-1.6, respectively, (p = NS). Fluid intake was similar between both groups (1875+/-1547 vs. 1506+/-970 ml, p = NS). Serum amylase was normal at baseline and remained virtually unchanged. Serum lipase was normal at baseline and immediately post-race in both groups, but increased at 48 hours post-race (82.2+/-34.3 to 121.5+/-53.3 mg/dl [A], p = 0.02 and 114.3+/-55.7 to 181.9+/-162.2 mg/dl [G], p = 0.09). CPK increased significantly and similarly in both groups immediately post-race, and even more dramatically 48 hours post-race (130.3+/-130.8 to 738.8+/-902.9, p = 0.007 to 1966.5+/-3.166.0 mg/dl [A] and 140.9+/-77.9 to 863.0+/-772.3, p = 0.003 to 5619+/-10636.8mg/dl [G]). Modest post-race decreases were seen in most serum amino acids in both groups. Finish times were longer than predicted (23+/-21 and 9+/-7 min for A and G groups, respectively, p = 0.049). Our study failed to show a clear benefit of arginine supplementation for the prevention of intestinal ischemia/reperfusion injury associated with endurance running, but either a detrimental affect on performance with arginine, or enhanced performance with glycine. Skeletal muscle injury was unaffected by arginine or glycine supplementation. The delayed increase in serum lipase suggests mild pancreatic injury, affected by either arginine or glycine supplementation.

We studied the effect of 9 h of simulated airplane cabin conditions at cruising altitude (8,000 feet; inspired oxygen equivalent to 15% O2 at sea level) on fetal plasma lactate in near-term pregnant rabbits. Controls (n = 19) spent 9 h at sea level (21% O2). Study group I (n = 21) experienced airplane cabin conditions. Study group II (n = 17) was studied at 8,000 feet with the inspired O2 concentration normalized to sea level. Study group III (n = 19) remained at sea level breathing 15% O2. Before ending each exposure, fetal blood sampling for lactate was performed under ultrasound guidance. Maternal lactates were obtained before and after sampling fetuses. Wilcoxon signed rank test, analysis of variance, and Bonferroni's method were used as appropriate. P < 0.05 denoted statistical significance. Study group I (altitude/hypoxia) had higher fetal lactates than controls (sea level/normoxia) and study group II (altitude/normoxia). Fetal lactates in study group I (altitude/hypoxia) were higher than in study group III (sea level/hypoxia). Maternal lactates were lower after fetal sampling. Fetal lactic acidemia was observed after 9 h of airplane cabin conditions. This was attributed to the combined effect of the lowered oxygen concentration and the decrease in atmospheric pressure.

Use of a real-time bedside glucose monitor was analyzed during the course of management of diabetic ketoacidosis (DKA) in children. Simultaneous determinations of blood glucose were obtained, using three methods: bedside glucose meter (One Touch II), laboratory glucose analyzer (YSI 2300 STAT), and a real-time bedside glucose monitor (VIA 1-01G Blood Chemistry monitor). Study patients included seventeen patients < 18 years of age admitted to a Pediatric Intensive Care Unit, with blood samples obtained during treatment of DKA by continuous insulin infusion. Four patients did not complete the study. Three experienced temporary technical problems with the monitor, and four required repeat IV placement. Duration of monitor use ranged between 6 and 47 h (mean 24 +/- 4 h). Blood glucose values ranged between 2.6 and 22.5 mmol/l. Overall correlation of blood glucose values were as follows: 0.965, 0.965, 0.973, VIA 1-01G vs. One Touch II, VIA 1-01G vs. YSI 2300 STAT, and One Touch II vs. YSI 2300 STAT, respectively (all P-values < 0.0001). This real-time bedside glucose monitor is accurate at glucose values < 13.8 mmol/l, and reliable for rapid, repetitive analyses. Results indicate that blood glucose values obtained using this real-time monitor are comparable to those using standard methods of measurement, and that this device is clinically applicable for use in management of children with DKA.

The alpha3 subunit of the neuronal nicotinic acetylcholine receptor is widely expressed in autonomic ganglia and in some parts of the brain. The alpha3 subunit can form heteromultimeric ion channels with other alpha subunits and with beta2 and beta4 subunits, but its function in vivo is poorly understood. We prepared a null mutation for the alpha3 gene by deletion of exon 5 and found that homozygous (-/-) mice lacked detectable mRNA on Northern blotting. The -/- mice survive to birth but have impaired growth and increased mortality before and after weaning. The -/- mice have extreme bladder enlargement, dribbling urination, bladder infection, urinary stones, and widely dilated ocular pupils that do not contract in response to light. Detailed histological studies of -/- mice revealed no significant abnormalities in brain or peripheral tissues except urinary bladder, where inflammation was prominent. Ganglion cells and axons were present in bladder and bowel. Bladder strips from -/- mice failed to contract in response to 0.1 mM nicotine, but did contract in response to electrical field stimulation or carbamoylcholine. The number of acetylcholine-activated single-channel currents was severely reduced in the neurons of superior cervical ganglia in -/- mice with five physiologically distinguishable nicotinic acetylcholine receptor subtypes with different conductance and kinetic properties in wild-type mice, all of which were reduced in -/- mice. The findings in the alpha3-null mice suggest that this subunit is an essential component of the nicotinic receptors mediating normal function of the autonomic nervous system. The phenotype in -/- mice may be similar to the rare human genetic disorder of megacystis-microcolon-intestinal hypoperistalsis syndrome.

Gastrointestinal complaints and occult bleeding have been commonly described in marathon runners. We hypothesized that these complaints may arise from intestinal ischemia caused by the shunting of blood away from the splanchnic circulation during endurance racing followed by reperfusion injury. Studies in animal models have suggested prophylactic vitamin E supplementation may prevent this type of injury. We sought to determine if prerace vitamin E supplementation would prevent intestinal ischemia/reperfusion injury in humans. Forty subjects who planned to complete the 1996 Houston-Tennaco Marathon were randomized to receive vitamin E (1000 IU daily) or placebo (soya lecithin) for 2 wk before the race in a double-blinded trial. Inclusion criteria included no use of non-steroidal anti-inflammatory drugs (NSAIDs) within 24 d of the race or vitamin or mineral supplements containing vitamins C or E or selenium within 30 d of the race. Subjects were studied 2 wk before the race and immediately following the race. Blood was obtained for serum vitamin E and total lipid and salicylate concentrations. A solution of lactulose (5 g) and mannitol (2 g) was consumed and urine was collected for 6 h. Aliquots were assayed for lactulose and mannitol concentration. Stool samples were tested for occult blood and following the race subjects rated their nausea, abdominal pain, and cramping on a 1-5 scale. Twenty-six subjects (24 male, 2 female) completed the marathon. Finish times ranged between 2 h 43 min and 5 h 28 min. All subjects had heme-negative stool prerace and four developed heme-positive stool postrace, with no difference between vitamin E and placebo groups (Fisher's exact = 0.63). All had non-detectable salicylate concentrations pre- and postrace. Serum vitamin E concentration increased in botPP = 0.02 in the vitamin E group and 1.45 +/- 0.40 to 1.66 +/- 0.48 mg/dL in the placebo group, P = 0.02). However, the serum vitamin E: total lipid ratio increased significantly in the vitamin E-supplemented group (0.0022 +/- 0.0002 to 0.0051 +/- 0.0015, P = 0.02), but not in the placebo group (P = 0.25). Overall, the urinary lactulose:mannitol ratio increased from 0.03 +/- 0.02 to 0.06 +/- 0.08 postrace (P = 0.06) without difference between vitamin E or placebo groups. Intestinal permeability increased significantly more in those who developed occult bleeding. More subjects in the placebo group developed abdominal cramping (Fisher's exact = 0.04) and abdominal pain (Fisher's exact = 0.06), although there was no difference in severity between groups. There was no difference in the incidence of nausea and no diarrhea was reported by any subject. Intestinal permeability tends to increase and occult gastrointestinal bleeding occurs during endurance running, suggesting the occurrence of intestinal ischemia/reperfusion injury. Prerace supplementation with the antioxidant vitamin E had no effect on performance, intestinal injury, occult bleeding, or the severity of postrace gastrointestinal complaints. Vitamin E supplementation was associated with a decreased incidence of these complaints but had no effect on their severity.

The concentrations of ferritin, transferrin and iron were measured in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) and control patients. Ferritin levels were significantly elevated in the CSF of chronic progressive active MS patients (4.71+/-0.54 ng/ml) compared to levels in normal individuals (3.07+/-0.17 ng/ml). MS patients with active or stable relapsing-remitting disease had ferritin levels that were comparable to those found in normal individuals. There were no significant differences in transferrin or iron levels in the CSF between MS and normal individuals. Both ferritin and transferrin levels were elevated in patients that had high CSF IgG values but not in patients with a high IgG index. Since ferritin binds iron, the increase of CSF ferritin levels in chronic progressive MS patients could be a defense mechanism to protect against iron induced oxidative injury. Ferritin levels could be a laboratory measure that helps to distinguish between chronic progressive and relapsing-remitting MS., (Copyright 1999 Elsevier Science B.V.)

Study Objectives: To determine the pharmacokinetic disposition of high doses of ibuprofen in patients with cystic fibrosis (CF), and to evaluate the reliability of intrapatient dosage adjustments to achieve recommended peak ibuprofen plasma concentrations., Design: First-order absorption, one-compartment model was fit to serial ibuprofen concentration-time data obtained from patients with CF and receiving high doses of ibuprofen 20-30 mg/kg., Setting: Medical school-affiliated teaching hospital., Patients: Ninety-eight patients with CF (53 males, 45 females; mean age 12.5 yrs)., Measurements and Main Results: The time to achieve apparent maximum ibuprofen concentration (Tmax) ranged from 1-3 hours, with maximum concentrations ranging from 21-150 microg/ml (mean 83 microg/ml). Apparent ibuprofen clearance (Cl/F) was significantly correlated with age (r2 = 0.43, p<0.0001) and measures of body size (body surface area [BSA] r2 = 0.50, p<0.0001). The Cl/F ranged from 21.1-114.7 ml/min/m2 (mean 45.5 ml/min/m2), a 5-fold difference. The Cl/F normalized to body weight decreased with increasing age (p=0.0009), but when normalized to BSA, there was no age-related change (p=0.65). Apparent volume of distribution was significantly correlated with age (r2 = 0.69, p<0.0001) and measures of body size (BSA r2 = 0.79, p<0.0001). Fourteen patients had ibuprofen dosage adjustments. The Cl/F was not different among doses; however, Tmax differed by an average of 1.25 hours (range 0-2 hrs)., Conclusion: The substantial variability in ibuprofen disposition and clearance we report is greater than previously described. Individualized dosages and therapeutic drug monitoring may be required to ensure plasma concentrations considered necessary to prevent pulmonary deterioration in patients with CF.

To examine the toxicity of cyclosiloxanes (CSs), the predominant low molecular weight cyclic silicones found in breast implants, we injected female CD-1 mice intraperitoneally with different doses of distillate (3.5-35 g/kg body weight) containing cyclosiloxane D3 (hexamethylcyclotrisiloxane; CS-D3), cyclosiloxane D4 (octamethylcyclotetrasiloxane; CS-D4), cyclosiloxane D5 (decamethylcyclopentasiloxane; CS-D5), and cyclosiloxane D6 (dodecamethylcyclohexasiloxane; CS-D6). The distillate was found to be lethal and all the mice injected with 35 g/kg died within 5-8 days. The median lethal dose (LD50) for distillate was estimated to be approximately 28 g/kg. These mice developed inflammatory lesions of the lung and liver as well as liver cell necrosis with elevated serum levels of alanine aminotransferase, aspartate aminotransferase, and lactic acid dehydrogenase. Administration of CS-D4 alone also produced lethality in these mice with an LD50 of 6-7 g/kg. CS-D4-treated mice also exhibited pulmonary and hepatic lesions and elevated serum enzymes. Analysis of LD50 data indicates that CS-D4 is about as toxic as carbon tetrachloride or trichloroethylene. We measured hydroxyl radical formation in CS-D4-treated mice and found increases of approximately 20-fold in liver and approximately 7-fold in lung on day 4 following injection. Our findings are significant because in vitro experiments have demonstrated that CSs can migrate out of breast implants, and in mouse experiments CSs have been shown to be widely distributed in many organs after a single subcutaneous injection and to persist for at least a year.

Objective: We sought to ascertain whether the timing of feeding initiation affected the development of intestinal lactase activity and whether there are clinical ramifications of lower lactase activity., Study Design: Preterm infants (26 to 30 weeks' gestation; n = 135) were randomly assigned to begin enteral feedings at either 4 (early group) or 15 days of age (standard group). At 10, 28, and 50 days of age lactase activity was determined by measuring the urinary ratio of lactulose/lactose after the 2 sugars were administered., Results: Lactase activity increased significantly over time. Infants in the early group had greater lactase activity at 10 days of age (by 100%) and 28 days of age (by 60%) than the standard group. At 10 days of age lactase activity was greater in milk- versus formula-fed infants. The time required to achieve full enteral feedings, the number of abnormal abdominal x-ray examinations, and the total number of abdominal x-ray examinations were inversely related to lactase activity., Conclusions: Early feeding increases intestinal lactase activity in preterm infants. Lactase activity is a marker of intestinal maturity and may influence clinical outcomes. Whether the effects of milk on lactase activity were due to the greater concentration of lactose in human milk compared with that in formula must be determined.