Randomized clinical trial of glutamine-supplemented versus standard parenteral nutrition in infants with surgical gastrointestinal disease.

BACKGROUND: Addition of glutamine to parenteral nutrition in surgical infants remains controversial. The aim of this trial was to determine whether glutamine supplementation of parenteral nutrition in infants requiring surgery would reduce the time to full enteral feeding and/or decrease the incidence of sepsis and septicaemia. METHODS: A prospective double-blind multicentre randomized clinical trial was performed in surgical infants less than 3 months old who required parenteral nutrition. Patients were allocated to treatment or control groups by means of minimization. Infants received either 0·6 g per kg per day alanyl-glutamine (treatment group) or isonitrogenous isocaloric parenteral nutrition (control group) until full enteral feeding was achieved. Primary outcomes were time to full enteral feeding and incidence of sepsis. Cox regression analysis was used to compare time to full enteral feeding, and to calculate risk of sepsis/septicaemia. RESULTS: A total of 174 patients were randomized, of whom 164 completed the trial and were analysed (82 in each group). There was no difference in time to full enteral feeding or time to first enteral feeding between groups, and supplementation with glutamine had no effect on the overall incidence of sepsis or septicaemia. However, during total parenteral nutrition (before the first enteral feed), glutamine administration was associated with a significantly decreased risk of developing sepsis (hazard ratio 0·33, 95 per cent confidence interval 0·15 to 0·72; P = 0·005). CONCLUSION: Glutamine supplementation during parenteral nutrition did not reduce the incidence of sepsis in surgical infants with gastrointestinal disease. REGISTRATION NUMBER: ISRCTN83168963 (http://www.controlled-trials.com).

Septicaemia due to enteric organisms is a later event in surgical infants requiring parenteral nutrition.

INTRODUCTION: The purpose of this study was to determine whether, in surgical infants requiring parenteral nutrition (PN), septicaemia due to enterococci or Gram-negative bacilli occurs later than septicaemia due to coagulase-negative staphylococci (CNS). PATIENTS/MATERIAL AND METHODS: We retrospectively studied 112 consecutive surgical infants (corrected gestational age up to 3 months) receiving PN for at least 5 days for congenital or acquired intestinal anomalies over a 2-year period (July 2007-June 2009). Data collected included diagnosis, duration of PN, episodes of septicaemia (defined as growth of bacteria from blood culture), and organisms cultured. We compared the time to first occurrence of septicaemia due to CNS with the times to first occurrence of septicaemia due to enterococci, Gram-negative bacilli, or other micro-organisms, using Kruskal-Wallis nonparametric ANOVA test and Dunn's multiple comparisons test. Data are given as median (range). RESULTS: 31 patients (28%) had a total of 65 episodes of septicaemia. Septicaemia due to CNS was most common, occurring in 22% of patients, after 17 days (1-239) of PN. Septicaemia due to enteric organisms was less common and occurred significantly later, at 59 (24-103) days for enterococci (p<0.01), and at 55 (30-106) days for Gram-negative bacilli (p<0.05). CONCLUSIONS: Septicaemia due to enterococci or Gram-negative bacilli occurs later in the course of PN than septicaemia due to CNS, in surgical infants. This suggests that these infants become more vulnerable to the translocation of enteric micro-organisms after a longer period of parenteral nutrition.

Review of the diagnoses predisposing infants to intestinal failure on hospitalized parenteral nutrition.

During the last 3 decades the use of parenteral nutrition (PN) and the aggressive introduction of enteral feeding in daily practice have transformed the outcome for even the sickest of these infants. More than 90% of infants and children now survive after extensive small bowel resection in the neonatal period. During the last 3 decades the use of parenteral nutrition (PN) and the aggressive introduction of enteral feeding in daily practice have transformed the outcome for even the sickest of these children. The aim of this study was to review the diagnoses (other than infants purely premature) that predispose infants to intestinal failure (IF) and dependency on PN as well as their outcomes. A total of 63 children less than 1 year old received PN for more than 28 days including 35 (56%) boys; 29% of cases were preterm infants with a median gestational age of 26.5 weeks (range, 24-33 weeks). The median age at the start of PN was 0.25 years or 3 months. Median duration of PN treatment was 62 days and median duration of hospitalization was 128 days. Twenty-three (36.5%) children had a primary nondigestive disorder (PNDD) and 40 (63.5%), a primary digestive disorder (PDD). Forty (63.5%) children with severe intestinal failure were successfully weaned off PN; whereas 8 (13%) infants with severe gastrointestinal diseases remained dependent on IV nutrition. Fourteen (22%) patients died. Infants less than 1 year of age with severe intestinal failure have up to a 75% survival rate, with a 65% chance of achieving intestinal autonomy. For children presenting with PDD in infancy, there is a high risk of needing long-term PN.

Sepsis and its etiology among hospitalized children less than 1 year of age with intestinal failure on parenteral nutrition.

Infections accompany intestinal failure (IF) more commonly in children than in adults, with reported incidences of 2% to 29%. Appropriate care of the central venous catheter is the most important factor preventing infections; but in addition, bacteria translocate from the dysmotile gut as a possible source of septicemia. The aim of this retrospective analysis was to investigate the rate and the epidemiologic profile of septicemia in the patient group at greatest risk, namely, children less than 1 year of age with IF on parenteral nutrition (PN). Among 63 children less than 1 year of age who were included over a 2-year period, 55% were boys. The overall median age at the start of PN was 0.3 years, with a mean duration of 80 days. Some 68% of patients had at least one episode of septicemia, experiencing a mean of 1.5 episodes (range, 1-12). Also, 19% of children displayed polymicrobial bloodstream infections. The most common Gram-positive pathogens were Staphylococcus spp and Enterococcus spp; the Gram-negative pathogens were Klebsiella spp followed by Enterobacter spp and E. coli. Infants less than 1 year of age with IF >28 days experienced a high (68%) rate of sepsis. There was no difference in the incidence of catheter-related blood stream infection according to the primary underlying diagnosis. The most common pathogens were Staphylococcus spp and Enterococcus spp, similar to etiologies of sepsis among children in intensive care units.

Disposition and tissue distribution of boron after infusion of borocaptate sodium in patients with malignant brain tumors.

PURPOSE: In the frame of the Czech boron neutron capture therapy (BNCT) project, a clinical Phase I study of borocaptate sodium [Na2B12H11SH (BSH)] as the boron-10 delivery agent was performed to obtain data on disposition and tissue distribution of boron after an infusion of this compound, as well as to establish an optimal protocol for BNCT of malignant cerebral tumors. METHODS AND MATERIALS: The kinetics of boron disposition after an infusion of borocaptate sodium (25 mg/kg body wt over the period of 1 h) was studied in a group of 10 patients with astrocytoma or glioblastoma of cerebral hemispheres using a modification of the Soloway-Messer colorimetric method. The boron content of tissues (tumor, healthy brain, dura mater, muscle, skin, and cranial bone) removed during the operation performed with latencies varying between 3 and 18 h was investigated by atomic emission spectrometry. RESULTS: Compartmental analysis of boron blood concentrations has shown that in the majority of patients (four males and three females), the concentration decline can be adequately described by a two-compartment pharmacokinetic model (i.e., by a biexponential relationship). The calculated half-lives of the initial (fast) phase of the concentration decline varied between 0.85 and 3.65 h, whereas the half-life values for the terminal (slow) phase ranged between 22.2 and 111.8 h. However, in the remaining three patients (all females), the goodness of fit of the boron concentration data was significantly better when a pharmacokinetic model with three compartments was assumed. In these patients, therefore, an additional ultrafast phase with a half-life varying between 17 and 37 min was detected in the beginning of the boron blood concentration decline. On the other hand, in one of these patients, the half-life of the terminal phase was found to be 415 h (i.e., more than 17 days). Such a long persistence in the body is explained by the very high value of the total distribution volume, indicating extensive binding of BSH in peripheral tissues. Another reason may be enterohepatic recycling of BSH. CONCLUSION: Tumor-to-blood ratios higher than 1.5, which are necessary for an effective outcome of BNCT, can be obtained only if the time interval elapsing between the onset of surgery and termination of BSH infusion is at least 12 h.

A histone deacetylase inhibitor potentiates retinoid receptor action in embryonal carcinoma cells.

Histone acetylation is thought to have a role in transcription. To gain insight into the role of histone acetylation in retinoid-dependent transcription, we studied the effects of trichostatin A (TSA), a specific inhibitor of histone deacetylase, on P19 embryonal carcinoma cells. We show that coaddition of TSA and retinoic acid (RA) markedly enhances neuronal differentiation in these cells, although TSA alone does not induce differentiation but causes extensive apoptosis. Consistent with the cooperative effect of TSA and RA, coaddition of the two agents synergistically enhanced transcription from stably integrated RA-responsive promoters. The transcriptional synergy by TSA and RA required the RA-responsive element and a functional retinoid X receptor (RXR)/retinoic acid receptor (RAR) heterodimer, both obligatory for RA-dependent transcription. Furthermore, TSA led to promoter activation by an RXR-selective ligand that was otherwise inactive in transcription. In addition, TSA enhanced transcription from a minimum basal promoter, independently of the RA-responsive element. Finally, we show that TSA alone or in combination with RA increases in vivo endonuclease sensitivity within the RA-responsive promoter, suggesting that TSA treatment might alter a local chromatin environment to enhance RXR/RAR heterodimer action. Thus, these results indicate that histone acetylation influences activity of the heterodimer, which is in line with the observed interaction between the RXR/RAR heterodimer and a histone acetylase presented elsewhere.

Retinoid X receptor (RXR) within the RXR-retinoic acid receptor heterodimer binds its ligand and enhances retinoid-dependent gene expression.

Retinoic acid receptor (RAR) and retinoid X receptor (RXR) form heterodimers and regulate retinoid-mediated gene expression. We studied binding of RXR- and RAR-selective ligands to the RXR-RAR heterodimer and subsequent transcription. In limited proteolysis analyses, both RXR and RAR in the heterodimer bound their respective ligands and underwent a conformational change in the presence of a retinoic acid-responsive element. In reporter analyses, the RAR ligand (but not the RXR ligand), when added singly, activated transcription, but coaddition of the two ligands led to synergistic activation of transcription. This activation required the AF-2 domain of both RXR and RAR. Genomic footprinting analysis was performed with P19 embryonal carcinoma cells, in which transcription of the RARbeta gene is induced upon retinoid addition. Paralleling the reporter activation data, only the RAR ligand induced in vivo occupancy of the RARbeta2 promoter when added singly. However, at suboptimal concentrations of RAR ligand, coaddition of the RXR ligand increased the stability of promoter occupancy. Thus, liganded RXR and RAR both participate in transcription. Finally, when these ligands were tested for teratogenic effects on zebra fish and Xenopus embryos, we found that coadministration of the RXR and RAR ligands caused more severe abnormalities in these embryos than either ligand alone, providing biological support for the synergistic action of the two ligands.

Kidney function changes in rats after single-dose administration of borocaptate sodium.

Kidney function changes after single-dose administration of borocaptate sodium (mercaptoundecahydro-closododecaborate, B12H11SH, BSH) were studied in rats. Changes of glomerular filtration rate (GFR) measured as 14C-inulin clearance, renal plasma flow rate (3H-p-aminohippuric acid clearance) and urine flow rate (UFR) after a slow intravenous injection of BSH (25 mg/kg b.w.) were investigated in rats under pentobarbital anaesthesia. It was found that a slow BSH injection induces a gradual decrease of renal plasma flow and glomerular filtration rate resulting in an almost constant reduction of the filtration fraction. These alterations were accompanied by a temporary increase of urine flow rate. Although a direct effect of BSH on the nephron cannot be excluded, it is suggested that the observed changes in kidney function might at least partly be mediated by disturbances in the function of the cardiovascular system following BSH injection. The role of the dianionic sulfhydryl group present in the borocaptate molecule in inducing these renal functional changes is discussed.

The diuretic effect of borocaptate sodium in rats and in patients with brain tumors.

Kidney function changes after single-dose administration of borocaptate sodium were studied in rats and in patients with brain tumors. Changes of glomerular filtration rate (GFR) measured as 14C-inulin clearance and urine flow rate (UFR) after a slow intravenous injection of BSH (25 and 50 mg/kg b.w., respectively) were investigated in rats under pentobarbital anesthesia. The effect of BSH has been compared with that of its disulfide (BSSB) which is spontaneously generated by oxidation of BSH during storage. It was found that BSH decreases GFR in relation to dose and, in the same way, causes a temporary increase of UFR. On the other hand, BSSB (50 mg/kg) induced a large reversible decrease of GFR as well as a decrease of urine excretion. Measurements of GFR (inulin clearance), renal plasma flow (PAH clearance) and urine excretion were taken in a group of patients with brain tumors in which boron disposition after an infusion of BSH (25 mg/kg b.w. over 1 h) had been studied. An increase in urine production was the dominant effect (up to 200% of the initial value), with the alterations of GFR and RPF being of minor significance except in one patient with a GFR reduction up to almost 50% the original value. Kidney function changes after BSH or BSSB administration are supposedly related to the high retention of BSH in kidney.

Successful surgical repair of aortic dissection presenting with complete heart block.

Acute dissection of the ascending aorta can present with complete heart block if the dissecting hematoma involves the interatrial septum near the atrioventricular node. We report a case of acute type A dissection presenting with complete heart block treated with emergency grafting of the ascending aorta, aortic valve replacement, and coronary artery bypass grafting. The patient survived, although complete heart block persisted requiring permanent pacemaker implantation.

Response of rat salivary glands to mastication of pelleted vitamin A-deficient diet.

Interpretation of previous studies of the effects of hypovitaminosis. A on salivary glands is confounded by the atrophic effects of liquid or powdered diets. The purpose of this study was to reevaluate the effects of vitamin. A deficiency on the morphology and function of rat salivary glands using a pelleted diet that promotes physiological levels of masticatory stimulation. Profound vitamin A deficiency resulted in a marked decrease in stimulated parotid secretion. Histological evaluation demonstrated the development of squamous metaplasia in the ducts of parotid, submandibular and sublingual salivary glands; however, atrophy was observed only in serious salivary glands. In the parotid gland the degree of atrophy corresponded to the decrease in stimulated secretion. Mild hypovitaminosis A (before the development of squamous metaplasia in ducts) was associated with distinctly different effects. The parotid gland was moderately enlarged. There was also a significant increase in stimulated secretion, which was not explained by changes in gland size, muscarinic receptor number or affinity, or receptor-mediated calcium signalling.

Loss of overproduction of polypeptide release factor 3 influences expression of the tryptophanase operon of Escherichia coli.

Expression of the tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and by tryptophan-induced inhibition of Rho-mediated transcription termination. Previous studies indicated that tryptophan induction might involve leader peptide inhibition of ribosome release at the stop codon of tnaC, the coding region for the operon-specified leader peptide. In this study we examined tna operon expression in strains in which the structural gene for protein release factor 3, prfC, is either disrupted or overexpressed. We find that prfC inactivation leads to a two- to threefold increase in basal expression of the tna operon and a slight increase in induced expression. Overexpression of prfC has the opposite effect and reduces both basal and induced expression. These effects occur in the presence of glucose and cyclic AMP, and thus Rho-dependent termination rather than catabolite repression appears to be the event influenced by the prfC alterations. prfC inactivation also leads to an increase in basal tna operon expression in various rho and rpoB mutants but not in a particular rho mutant in which the basal level of expression is very high. The effect of prfC inactivation was examined in a variety of mutants with alterations in the tna leader region. Our results suggest that translation of tnaC is essential for the prfC effect. The tryptophan residue specified by tnaC codon 12, which is essential for induction, when replaced by another amino) acid, allows the prfC effect. Introducing UAG or UAA stop codons rather than the normal tnaC UGA stop codon, in a strain with an inactive prfC gene, also leads to an increase in the basal level of expression. Addition of the drug bicyclomycin increases basal operon expression of all mutant strains except a strain with a tnaC'-'lacZ fusion. Expression in the latter strain is unaffected by prfC alterations. Our findings are consistent with the interpretation that ribosome release at the tnaC stop codon can influence tna operon expression.

RAR and RXR selective ligands cooperatively induce apoptosis and neuronal differentiation in P19 embryonal carcinoma cells.

Retinoids cause differentiation in embryonal carcinoma (EC) cells, thus mimicking events in mammalian development. Here, we show that retinoids also cause apoptosis in P19 EC cells. Characteristic DNA fragmentation was observed within 36 h after addition of retinoic acid (RA). Synthetic retinoids that are selective for RA receptors (RAR) were also effective in inducing apoptosis, whereas RXR selective ligands were without effect. The combination of RAR and RXR ligands resulted in a synergistic increase in apoptotic cell death. As with apoptosis, neuronal differentiation of P19 cells was synergistically induced by the combination of RAR and RXR ligands. Data obtained with an RAR antagonist and with P19 cells carrying a dominant negative RXR indicate that the two processes are receptor mediated. Together, our results indicate that retinoid-induced apoptosis and neuronal differentiation are closely coupled, and that both RAR and RXR play a role in these processes as active receptors for their respective ligands.

On the role of helix 0 of the tryptophan synthetase alpha chain of Escherichia coli.

The role of helix 0 of the alpha chain (TrpA) of the tryptophan synthetase alpha2beta2 multi-functional enzyme complex of Escherichia coli was examined by deleting amino-terminal residues 2-6, 2-11, or 2-19 of TrpA. Selected substitutions were also introduced at TrpA positions 2-6. The altered genes encoding these polypeptides were overexpressed from a foreign promoter on a multicopy plasmid and following insertion at their normal chromosomal location. Each deletion polypeptide was functional in vivo. However all appeared to be somewhat more labile and insoluble and less active enzymatically than wild type TrpA. The deletion polypeptides were overproduced and solubilized from cell debris by denaturation and refolding. Several were partially purified and assayed in various reactions in the presence of tryptophan synthetase beta2 (TrpB). The purified TrpADelta2-6 and TrpADelta2-11 deletion polypeptides had low activity in both the indole + serine --> tryptophan reaction and the indoleglycerol phosphate + serine --> tryptophan reaction. Poor activity in each reaction was partly due to reduced association of TrpA with TrpB. The addition of the TrpA ligands, alpha-glycerophosphate or indoleglycerol phosphate, during catalysis of the indole + serine --> tryptophan reaction increased association and activity. These findings suggest that removal of helix 0 of TrpA decreases TrpA-TrpB association as well as the activity of the TrpA active site. Alignment of the TrpA sequences from different species indicates that several lack part or all of helix 0. In some of these polypeptides, extra residues at the carboxyl end may substitute for helix 0.

Differential 9-cis-retinoic acid-dependent transcriptional activation by murine retinoid X receptor alpha (RXR alpha) and RXR beta. Role of cell type and RXR domains.

The 9-cis-retinoic acid (9cRA)-inducible enhancer of the rat cellular retinol-binding protein type II gene (CRBP II) was shown to be differentially regulated by the murine retinoid X receptor alpha (RXR alpha) as compared with RXR beta. Transient transfection assays performed in NIH 3T3 fibroblast cells demonstrated that RXR alpha yielded a high level of 9cRA-dependent transcription of a reporter gene linked to the CRBP II enhancer, when compared with RXR beta. This effect was cell type-dependent, since both receptors elicited comparable transcriptional activation of the same reporter in P19 embryonal carcinoma cells. To further explore the structural determinants responsible for the differences between these two receptors, a series of chimeric receptor constructs were made. Co-transfection assays utilizing these chimeras demonstrated that both the N terminus and the hinge region connecting the DNA binding domain with the ligand binding domain of RXR alpha were responsible for the high level of 9cRA-dependent transcription observed in NIH 3T3 cells, Furthermore, the hinge region of RXR alpha was shown to be necessary to repress, in the absence of hormone, the transcriptional activation function located in the N-terminal domain of RXR alpha. These results stress the importance of functional links between different RXR domains and suggest an RXR subtype and cell type-dependent specificity in the control of the 9cRA response.

Bicyclomycin sensitivity and resistance affect Rho factor-mediated transcription termination in the tna operon of Escherichia coli.

The growth-inhibiting drug bicyclomycin, known to be an inhibitor of Rho factor activity in Escherichia coli, was shown to increase basal level expression of the tryptophanase (tna) operon and to allow growth of a tryptophan auxotroph on indole. The drug also relieved polarity in the trp operon and permitted growth of a trp double nonsense mutant on indole. Nine bicyclomycin-resistant mutants were isolated and partially characterized. Recombination data and genetic and biochemical complementation analyses suggest that five have mutations that affect rho, three have mutations that affect rpoB, and one has a mutation that affects a third locus, near rpoB. Individual mutants showed decreased, normal, or increased basal-level expression of the tna operon. All but one of the resistant mutants displayed greatly increased tna operon expression when grown in the presence of bicyclomycin. The tna operon of the wild-type drug-sensitive parent was also shown to be highly expressed during growth with noninhibitory concentrations of bicyclomycin. These findings demonstrate that resistance to this drug may be required by mutations at any one of three loci, two of which appear to be rho and rpoB.

Triple-drug immunosuppression with steroid discontinuation by six months after heart transplantation.

BACKGROUND: Triple-drug immunosuppression with cyclosporine, azathioprine, and prednisone is associated with complications which might be reduced by steroid withdrawal. METHODS: In two groups of heart transplant recipients maintained on an identical regimen of cyclosporine and azathioprine, prednisone was withdrawn in group I patients (n = 35) by 6 months after transplantation, whereas in group II patients (n = 49) prednisone was never discontinued. RESULTS: Survival was similar in the two groups. The incidence of acute graft rejection was significantly higher in group I (54%) than in group II (12%), whereas infective complications were significantly lower in group I than in group II (0.63 versus 1.02 episode/patient). The degree of posttransplantation weight gain, lipid abnormalities, and incidence of hypertension were not modified by the fast tapering of prednisone, whereas the incidence of cataract and compression fracture and the degree of bone loss were significantly reduced in group I. Graft function and incidence of coronary artery disease were similar in the two groups. CONCLUSIONS: The present data suggest that prednisone can be safely withdrawn in heart transplant recipients without jeopardizing survival and graft function. Longer follow-up is needed to assess the full impact of early withdrawal of steroids from triple-drug immunosuppression, especially on long-term graft function and incidence of coronary artery disease. Benefits of early steroid withdrawal included a reduction in bone loss, which might ultimately have a major positive impact on the extent of long-term rehabilitation and exercise tolerance after heart transplantation.

The in vitro effect of sodium mercaptoundecahydrododecaborate (borocaptate) on sulfhydryl groups of different rat tissues.

The effect of borocaptate on structural protein sulfhydryl (SH) groups of different organs and kidney subcellular fractions was studied in vitro. It was shown that the binding capacity of borocaptate is not the same for different tissues and subcellular fractions. The highest binding capacity was in the liver, while kidney and brain values were significantly lower. Therefore, it appears that the same concentration of borocaptate may have different effects in various organs.

Role of regulatory features of the trp operon of Escherichia coli in mediating a response to a nutritional shift.

Physiological studies were performed under nutritional stress and nonstress conditions to assess the relative importance of the various regulatory mechanisms that Escherichia coli can use to alter its rate of tryptophan synthesis. Mutants were examined in which the trp repressor was inactive, transcription termination at the trp attenuator was altered, transcription initiation at the trp promoter was reduced, or feedback inhibition of anthranilate synthase was abolished. Strains were examined in media with and without tryptophan, phenylalanine and tyrosine, or acid-hydrolyzed casein and following shifts from one medium to another. Growth rates and anthranilate synthase levels were measured. In media lacking tryptophan, each of the mutants showed relief of repression and/or attenuation and maintained a near-normal growth rate. Following a shift from a medium containing tryptophan to a tryptophan-free medium containing phenylalanine and tyrosine or acid-hydrolyzed casein, mutants with abnormally low trp enzyme levels exhibited an appreciable growth lag before resuming growth. The wild-type strain displayed termination relief only under one extreme shift condition, upon transfer from a minimal medium containing tryptophan to minimal medium with only phenylalanine and tyrosine. A promoter down-mutant had difficulty adjusting to a shift from high tryptophan to low tryptophan levels in a medium containing acid-hydrolyzed casein. In all media tested, anthranilate synthase levels were lower in a feedback-resistant mutant than in the wild type. These studies demonstrate the capacity of E. coli to adjust its rate of tryptophan synthesis to maintain rapid growth following a shift to stressful nutritional conditions.