[D-lactic acidosis in a child with short bowel syndrome]. / Acidose d-lactique chez un enfant présentant un syndrome de grêle court.

INTRODUCTION: d-lactic acidosis is a rare and severe complication of short bowel syndrome in children that may result from important ileal bacterial overgrowth by lactobacilli. Intestinal flora (Lactobacilli) is responsible for the production of d-lactic acid after fermentation of food carbohydrates. OBSERVATION: We report on the case of a 6-year-old child with a short bowel syndrome treated with both home enteral and parenteral nutrition. The patient suddenly presented with acute neurological symptoms including dysarthria and disorientation. Biological analysis revealed metabolic acidosis, increased plasma d-lactic acid assessed by organic acid chromatography analysis and a very important increase in expired hydrogen during glucose breath test. Lactobacillus fermentum (known to produce d and L isomers of lactic acid) was isolated in the gastric liquid and rectal swabs. Clinical and biological evolution was rapidly favourable after treatment with intravenous sodium bicarbonate, antibiotic therapy and interruption of enteral nutrition. CONCLUSION: d-lactic acidosis should be suspected when neurological symptoms occur in a child with short bowel syndrome. They can be prevented by treating intestinal bacterial overgrowth.

Efficacy of methotrexate in pediatric Crohn's disease: a French multicenter study.

BACKGROUND: Immunosuppressors play a major role in maintaining remission in Crohn's disease (CD). In patients who do not tolerate or escape therapy with azathioprine (AZA)/6-mercaptopurine, there is a marked need for other immunosuppressive drugs. The aim of the present study was to evaluate the efficacy and safety of methotrexate (MTX) in children with active CD. METHODS: In a retrospective multicenter (n = 3) study, the efficacy of MTX to induce complete remission or a clinical improvement was analyzed in 61 children with active CD. RESULTS: CD was diagnosed at a mean age of 11.1 +/- 2.3 years, and MTX was introduced 3.1 +/- 2.2 years after diagnosis. Indications to use MTX were a nonresponse to or relapse under AZA (n = 42) or AZA intolerance/toxicity (n = 19). MTX improved or induced complete remission in 49 patients (80%), of whom 18 (29.5%) relapsed after 13 +/- 10 months of treatment. Under MTX medication, complete remission was observed in 39%, 49%, and 45% at 3, 6, and 12 months, respectively. Follow-up over at least 24 months in 11 children confirmed a sustained remission on MTX monotherapy up to 40 months. Adverse reactions were observed in 14 patients (24%), requiring discontinuation of MTX in 6 children (10%) (liver enzyme elevation, n = 2; varicella-zoster, n = 1; nausea, n = 3). MTX allowed corticosteroid discontinuation in 36 patients. CONCLUSIONS: MTX improved the clinical course in most pediatric CD patients who escaped or did not tolerate AZA. Short-time toxicity of MTX resulted in drug discontinuation in <10%. These data point to a beneficial and safe use of MTX in the treatment of pediatric CD.

Mitomycin C: an alternative conservative treatment for refractory esophageal stricture in children?

BACKGROUND AND STUDY AIMS: Mitomycin C is an antiproliferative agent that has been used successfully as an adjunct treatment in ophthalmological procedures, in the management of laryngeal and tracheal stenosis, and more recently to prevent the recurrence of caustic esophageal strictures in children. The aim of this study was to assess the efficacy and safety of local application of mitomycin C to refractory esophageal strictures in children. PATIENTS AND METHODS: We performed a preliminary prospective study of local application of mitomycin C in four children, aged between 1 year and 6 years, who had refractory esophageal strictures. Two of the children presented with strictures caused by caustic ingestion and the other two children had anastomotic strictures following surgical repair of congenital esophageal atresia. The patients had required between four and ten esophageal dilations over a 5-24-month period before mitomycin C application. After an endoscopic dilation, mitomycin C was applied onto the dilation wound using a rigid endoscope. RESULTS: No complications were observed after the procedure. One child required a second application of mitomycin C 2 weeks after the first application because of recurrence of dysphagia. All the children remained asymptomatic and none of them required further dilation over a mean follow-up period of 24 months. Radiological control examinations revealed that there was no recurrence of the esophageal strictures and esophageal biopsies performed during follow-up showed no signs of dysplasia. CONCLUSIONS: Local application of mitomycin C is a potential alternative to iterative dilations, surgery, or stent placement for the treatment of refractory esophageal strictures in children. However, prospective, long-term assessment of outcomes is needed before any definitive conclusion can be drawn about the usefulness of mitomycin C in these patients.

[Treatment of acute diarrhea: prescription patterns by private practice pediatricians]. / Traitement des diarrhées aiguës: les habitudes de prescription des pédiatres libéraux.

UNLABELLED: Acute gastroenteritis remains a frequent illness in infants and children with still important morbidity and mortality rates. Oral rehydratation solutions (ORS) and early refeeding are the main recommendations. Indication of drugs remains limited. OBJECTIVE: To evaluate the management of acute diarrhea by private practice pediatricians of France. METHODS: A questionnaire concerning ORS, dietary formula, antidiarrheal diet, antibiotherapy, antidiarrheal drugs was sent to all 2907 private pediatricians of France. RESULTS: Six hundred twenty-nine questionnaires were analyzed (22%). Three hundred and ninety-seven pediatricians (63%) prescribed systematically an ORS, 294 (47%) changed formula, 412 (66%) prescribed a regimen. Antibiotic was prescribed after coproculture (81%), when glairy and bloody diarrhea (65%), associated infectious disease (63%), toxi-infectious syndrome (42%) or immunodeficiency were present (28%). Most pediatricians (97%) prescribed at least one drug: diosmectite (84%), Lactobacillus acidophilus (63%), Saccharomyces boulardii (62%), racecadotril (62%), loperamide (28%), attapulgite de Mormoiron (26%), nifuroxazide (20%). Drugs were prescribed more often for their effectiveness than for comfort. CONCLUSION: This study demonstrates the discrepancies that remain between recommendations and practical care in the treatment of acute diarrhea in children. Private French pediatricians often prescribe drugs.

Characterization of alpha(1)-adrenoceptor subtypes in the eye.

The presence of the alpha(1)-adrenoceptor subtypes in various parts of the pig and rabbit eyes was investigated using [(3)H]-prazosin radioligand binding. The characterization of the subtypes was achieved by performing competition experiments with various subtype selective drugs. In the pig retina, both alpha(1A)- and alpha(1B)-adrenoceptors were detected and the proportion of sites was 70% alpha(1A)- and 30% alpha(1B)-adrenoceptors, respectively. In the pig iris, ciliary body and choroid, which are melanin-rich tissues, the non-specific binding of [(3)H]-prazosin was too high to detect any of the alpha(1)-adrenoceptor subtypes. However, in the albino rabbit iris, ciliary body and retina both alpha(1A)- and alpha(1B)-adrenoceptors were detected. The proportion of sites in the iris was 60 % alpha(1A)- and 40% alpha(1B)-adrenoceptors, respectively. In the ciliary body and rabbit retina the proportion of sites were 70% alpha(1A)- and 30% alpha(1B)-adrenoceptors. Only the alpha(1A)-adrenoceptor subtype was detected in the rabbit choroid.

Specific binding of proinsulin C-peptide to human cell membranes.

Recent reports have demonstrated beneficial effects of proinsulin C-peptide in the diabetic state, including improvements of kidney and nerve function. To examine the background to these effects, C-peptide binding to cell membranes has been studied by using fluorescence correlation spectroscopy. Measurements of ligand-membrane interactions at single-molecule detection sensitivity in 0.2-fl confocal volume elements show specific binding of fluorescently labeled C-peptide to several human cell types. Full saturation of the C-peptide binding to the cell surface is obtained at low nanomolar concentrations. Scatchard analysis of binding to renal tubular cells indicates the existence of a high-affinity binding process with K(ass) > 3.3 x 10(9) M(-1). Addition of excess unlabeled C-peptide is accompanied by competitive displacement, yielding a dissociation rate constant of 4.5 x 10(-4) s(-1). The C-terminal pentapeptide also displaces C-peptide bound to cell membranes, indicating that the binding occurs at this segment of the ligand. Nonnative D-C-peptide and a randomly scrambled C-peptide do not compete for binding with the labeled C-peptide, nor were crossreactions observed with insulin, insulin-like growth factor (IGF)-I, IGF-II, or proinsulin. Pretreatment of cells with pertussis toxin, known to modify receptor-coupled G proteins, abolishes the binding. It is concluded that C-peptide binds to specific G protein-coupled receptors on human cell membranes, thus providing a molecular basis for its biological effects.

Characterization of the enzymatic activity for biphasic competition by guanoxabenz (1-(2,6-dichlorobenzylidene-amino)-3-hydroxyguanidine) at alpha2-adrenoceptors. II. Description of a xanthine-dependent enzymatic activity in spleen cytosol.

The mechanism for formation of high affinity binding of guanoxabenz (1-(2,6-dichlorobenzylidene-amino)-3-hydroxyguanidine) to alpha2-adrenoceptors by the rat spleen cytosol was studied. We report here that the spleen cytosolic fraction mediated the reduction of guanoxabenz to guanabenz (1-(2,6-dichlorobenzylidene-amino)-3-guanidine), the latter having an almost 100-fold higher affinity for rat alpha2A-adrenoceptors than guanoxabenz itself. The reaction product could be separated by high-performance liquid chromatography and its identity as guanabenz confirmed by nuclear magnetic resonance. The spleen cytosolic activity could be separated into high and low molecular weight components, the high molecular weight component requiring low molecular weight factors for maximal activity. Xanthine oxidase seems to be the most likely candidate responsible for the activity, as the guanoxabenz-reducing activity of the high molecular weight component could be sustained by exogenously applied xanthine, while it was potently blocked by allopurinol. The conversion of guanoxabenz by the cytosolic activity was also quite potently blocked by DWO1, 1-(3,4-dimethoxybenzylideneamino)3-hydroxyguanidine, a hydroxyguanidine analogue to guanoxabenz.

Characterization of the enzymatic activity for biphasic competition by guanoxabenz (1-(2,6-dichlorobenzylidene-amino)-3-hydroxyguanidine) at alpha2-adrenoceptors. I. Description of an enzymatic activity in spleen membranes.

The mechanism for formation of high-affinity binding of 1-(2,6-dichlorobenzylidene-amino)-3-hydroxyguanidine (guanoxabenz) to alpha2-adrenoceptors was studied in particulate fractions from the rat spleen. The proportion of apparent high versus low-affinity alpha2-adrenoceptor binding sites increased with increasing incubation time and was also augmented by Mg2+ ions. The formation of high-affinity guanoxabenz binding seemed to be inhibited by a series of N-hydroxyguanidine analogs to guanoxabenz, as well as by a series of metabolic inhibitors that included allopurinol, 1-chloro-2,4-dinitrobenzene, 5,5'-dithiobis-(2-nitrobenzoic acid), cibacron blue, phenyl-p-benzoquinone, didox, and trimidox. The formation of guanoxabenz high-affinity binding was also inhibited in a time- and concentration-dependent fashion by preincubating the membranes with the LW03 N-hydroxyguanidine analogue of guanoxabenz. Moreover, when the spleen membranes were extensively washed for 30 min with buffers at 25 degrees, the guanoxabenz high-affinity binding disappeared. However, when these washed membranes were supplemented with xanthine, the apparent affinity of guanoxabenz increased four to five-fold. Taken together, all data were compatible with the theory that the formation of high-affinity binding was dependent on the generation of a guanoxabenz metabolite that showed an approximate 100-fold greater affinity for the alpha2-adrenoceptors than guanoxabenz itself. Because the most potent blocker of the formation of high-affinity binding was allopurinol (apart from some N-hydroxyguanidine analogs to guanoxabenz) and since the activity could be restored with xanthine, a likely candidate responsible for the metabolic activation is xanthine oxidase.

Conditions for biphasic competition curves in radioligand binding for ligands subjected to metabolic transformation.

The conditions affording biphasic competition curves in radioligand binding for ligands subjected to metabolic transformation was analyzed theoretically. It was shown that when a competing ligand was subjected to transformation to a ligand that showed higher affinity than the parent compound, biphasic competition curves, which might wrongly be interpreted as indicating the presence of two receptor sites, could be observed in binding assays containing a homogenous receptor population. Biphasic competition curves were seen if the conversion of the competitor occurred according to zero and second order kinetics, as well as by enzymatic catalytic processes. However, when the conversion occurred according to a first order kinetics, the competition curves were uniphasic and resolved only into one-site fits, with the apparent affinity of the competitor reflecting the degree of conversion of the competitor to its metabolite. When the metabolic conversion resulted in a metabolite that showed lower affinity for the receptor than the parent compound, the competition curves became supersteep for conversions according to zero and second order kinetics, as well as for conversion by enzymatic catalytic processes.

Identification of an N-hydroxyguanidine reducing activity of xanthine oxidase.

A guanoxabenz [1-(2,6-dichlorobenzylideneamino)-3-hydroxyguanidine; an N-hydroxyguanidine] reducing enzymatic activity of rat spleen cytosol was investigated. By means of protein purification and N-terminal amino acid sequencing, the reducing activity was shown to reside in xanthine oxidase. The action of the enzyme on guanoxabenz resulted in the formation of guanabenz [1-(2,6-dichlorobenzylidene-amino)-3-guanidine]; the product formation could be monitored by HPLC and its identity was confirmed by NMR analysis. The reduction of guanoxabenz required xanthine or NADH as reducing substrates, while the process could be blocked by allopurinol, a selective inhibitor of xanthine oxidase. By using bovine milk xanthine oxidase, the guanoxabenz reducing activity of the enzyme was also verified. We conclude that guanoxabenz is a novel electron acceptor structure for xanthine oxidase.

Characterization of guanoxabenz reducing activity in rat brain.

Guanoxabenz (1-(2,6-dichlorobenzylidene-amino)-3-hydroxyguanidine) and guanabenz (1-(2,6-dichlorobenzylidene-amino)-3-guanidine) are both known as centrally active antihypertensive drugs. We have previously shown that enzymatic activity in the rat spleen can induce N-reduction of guanoxabenz, leading to high affinity alpha 2-adrenoceptor binding, due to the formation of the alpha 2-adrenoceptor active drug, guanabenz. The spleen activity appears to reside in xanthine oxidase as it is activated by xanthine and blocked by allopurinol. We report that high affinity guanoxabenz binding is also induced in rat brain membranes after addition of NADH or NADPH cofactors. However, the brain process was clearly different from that of the spleen, as the formation of high affinity binding in the brain was not blocked by allopurinol. Moreover the NADH/NADPH activated mechanism of the brain membranes was not blocked by carbon monoxide and SKF525A, thus the activity appears not to reside in cytochrome P450 enzymes. Instead the activity was blocked by menadione and dicumarol. We conclude that the rat cerebral cortex contains an enzymatic activity that may activate guanoxabenz leading to formation of a metabolite showing high affinity for alpha 2-adrenoceptors. We also conclude that the rat brain activity is clearly distinct from that of the rat spleen.

Characterization of alpha1-adrenoceptor subtypes in the pig.

The identities of the alpha1-adrenoceptor subtypes present in various tissues of the pig were studied using [3H]prazosin radioligand binding. The subtypes were characterized by performing competition experiments for various subtype selective drugs. In the cerebral cortex, spleen and heart, both alpha1A- and alpha1B-adrenoceptors were detected. In the liver was found only the alpha1A-subtype, while in the aorta was found only the alpha1B-subtype. An alpha1-adrenoceptor subtype was present in the adrenal gland with a high affinity for prazosin, the pKd value being 9.6, but with relatively low affinities for other alpha1-adrenoceptor binding drugs. The adrenal gland alpha1-adrenoceptor did not seem to represent the classical alpha1D-subtype, since drugs selective for the alpha1D-subtype in other species, including BMY7378 and SKF104856, showed low affinities for the pig adrenal gland alpha1-adrenoceptor.

Localization and changes in distribution of brain alpha 2- and beta-adrenoceptors in response to acclimation state in the American bullfrog (Rana catesbeiana).

Alpha (alpha)- and beta (beta)-adrenoceptors regulate physiological processes in vertebrates. This study determined the location of alpha 2- and beta-adrenoceptors in the brain of the American bullfrog, Rana catesbeiana, using autoradiography. As the density of receptors may be affected by environmental temperature, a comparative numerical analysis of adrenoceptors in the areas of localization with respect to warm and cold acclimation was also carried out. Areas of greatest concentration of alpha 2-adrenoceptors were the accessory olfactory bulb, medial pallium, and olfactory bulb. Adrenoceptor numbers were significantly decreased in the accessory olfactory bulb and medial pallium in cold-acclimated animals. beta-adrenoceptors were localized in the thalamus, cerebellum, medial pallium, and amygdala/ striatum. Cold acclimation decreased adrenoceptor density in medial pallium and torus semicircularis and increased adrenoceptor density in the thalamus and hypothalamic preoptic areas. Among the alpha 2- and beta-adrenoceptors, only four regions of overlap existed, the medial pallium, hypothalamic preoptic area, optic tract, and isthmic tegmentum. Otherwise, where there were alpha 2-adrenoceptors, there were few or no beta-adrenoceptors. No alpha 2- or beta-adrenoceptors were found in the pituitary and optic chiasm. The distribution of adrenoceptors in particular areas of the brain may have functional significance with respect to physiological changes which occur in response to hibernation.

[3H]RS79948-197 binding to human, rat, guinea pig and pig alpha2A-, alpha2B- and alpha2C-adrenoceptors. Comparison with MK912, RX821002, rauwolscine and yohimbine.

The Kd values of the recently introduced radioligand [3H]RS79948-197 ((8a R,12aS,13a-S)-5,8,8a,9,10,11,12,12a,13,13a-decahydro-3-metho xy-12-(ethylsulphonyl)-6H-isoquino[2,1-g][1,6]naphthyridine) were determined for the recombinant human and rat alpha2A-, alpha2B- and alpha2C- as well as guinea pig alpha2B- and alpha2c-adrenoceptors expressed in COS (CV-1 Origin, SV40) cells. In addition, the Kd values were also determined for [3H]RS79948-197 for the guinea pig spleen alpha2A-adrenoceptor and for pig alpha2A-, alpha2B- and alpha2C-adrenoceptors in membranes obtained from kidney and striatum. Available radioligands for alpha2-adrenoceptors, besides [3H]RS79948-197 are the tritiated forms of MK912 ((2S,12bS)1',3'-dimethylspiro(1,3,4,5',6,6',7,12b-octa hydro-2H-benzo[b]furo[2,3-a]quinazoline)-2,4'-pyrimidin-2'-one), RX821002 (2-methoxy-idazoxan), rauwolscine and yohimbine. In the present article the binding constants of all these substances for the alpha2A-, alpha2B- and alpha2C-adrenoceptor subtypes in human, pig, rat and guinea pig are reviewed. In all species tested MK912 was alpha2C-selective, RX821002 showed a minor alpha2A-selectivity, whereas [3H]RS79948-197 was non-selective among the alpha2-adrenoceptor subtypes, showing high affinity for all three subtypes. Rauwolscine and yohimbine showed relatively low affinities for nmost of the alpha2-adrenoceptor subtypes investigated, the exception being rauwolscine having high affinity for the human and porcine alpha2C-adrenoceptors.

Quantification of alpha2A and alpha2C adrenoceptors in the rat striatum and in different regions of the spinal cord.

The alpha2C-adrenoceptor preferring radioligand [3H]-MK912 was used for labelling alpha2A- and alpha2C-adrenoceptors in the rat striatum, in the cervical, thoracic and lumbar parts of the spinal cord, and in the dorsal and ventral halves of the spinal cord. In addition, guanfacine was used as a tool to delineate the alpha2A- and alpha2C-adrenoceptors. In the striatum the sites were 72% alpha2A- and 28% alpha2C-adrenoceptors, while in all regions of the spinal cord the proportions of the sites were about 96% alpha2A- and 4% alpha2C-adrenoceptors. A multi-curve experimental design and computer analysis was used in order to enable the accurate quantification of the alpha2A- and alpha2C-adrenoceptors in the striatum and spinal cord.

Autoradiographic studies of central alpha 2A- and alpha 2C-adrenoceptors in the rat using [3H]MK912 and subtype-selective drugs.

In the present study we examined the distribution of alpha 2A- and alpha 2C-adrenoceptors in tissue slices from the rat cervical spinal cord and from brain slices collected at the level of the striatum. To differentiate between alpha 2A- and alpha 2C-adrenoceptors, the slices were incubated with [3H]MK912 in the presence of graded concentrations of the alpha 2A-selective drug, BRL44408, or the alpha 2C-selective drug, spiroxatrine. Computer analysis of the autoradiograms indicated that 0.4 nM [3H]MK912 plus 185 nM BRL44408 selectively labeled alpha 2C-adrenoceptors, while 0.4 nM [3H]MK912 plus 220 nM spiroxatrine selectively labeled alpha 2A-adrenoceptors. Using this approach, alpha 2C-adrenoceptors were detected in the striatum, while alpha 2A-adrenoceptors predominated in the cortical layers 1-4, the spinal cord distal dorsal horn, the septum and the endopiriform nucleus.

Characterization of adrenoceptor types and subtypes in American bullfrogs acclimated to warm or cold temperature.

While indirect evidence suggested that the responsiveness of frog adrenoceptors changes in response to temperature, direct measurement of adrenoceptor binding following acclimation to warm and cold temperatures had not been done. In the present study, the radioligands [3H]prazosin, [3H]RX821002, and [125I]cyanopindolol were used to label and quantify alpha 1-, alpha 2-, and beta-adrenoceptors in bullfrogs acclimated to warm or cold environments. The number of alpha 1-, alpha 2-, and beta-adrenoceptors in atrium, ventricle, and kidney membranes was not significantly different between warm- and cold-acclimated frogs. Characterization of receptor subtypes using pharmacological antagonists demonstrated that alpha 2-adrenoceptors in frog spinal cord and kidney were of the same pharmacological subtype, which is similar to the mammalian alpha 2A-subtype. The beta-adrenoceptor in frog ventricle, atrium, and kidney was the beta 2-subtype. These results suggest that while the alpha 1-, alpha 2-, and beta-adrenoceptor types have evolved in the frog, multiple subtypes of adrenoceptors are not necessary for physiological regulation in this species.

Characterization of alpha 2-adrenoceptor subtypes in the porcine eye: identification of alpha 2A-adrenoceptors in the choroid, ciliary body and iris, and alpha 2A- and alpha 2C-adrenoceptors in the retina.

The subtypes of alpha 2-adrenoceptor were characterized in the choroid, ciliary body, iris and retina of the pig eye by using radioligand binding. [3H]-MK912 labelled dense populations of alpha 2A-adrenoceptors in the choroid and ciliary body. In the retina, [3H]-MK912 labelled both alpha 2A- and alpha 2C-adrenoceptors. In the iris, receptors of the alpha 2A-adrenoceptor type were detected by using either [3H]-MK912 or [3H]-RX821002 as radioligands.

Identification of drugs subtype-selective for alpha 2A-, alpha 2B-, and alpha 2C-adrenoceptors in the pig cerebellum and kidney cortex.

The radioligands [3H]MK912 and [3H]RX821002 were used to label alpha2A-, alpha2B-, and alpha2C-adrenoceptors of the pig cerebellum and kidney cortex. By inclusion of the alpha2A-adrenoceptor-selective drug, BRL44408, and using a 'multi-curve' experimental design all the three porcine alpha2-adrenoceptor subtypes could be characterized pharmacologically. The data indicate that the pig alpha2-adrenoceptor subtypes are pharmacologically more related to human alpha2-adrenoceptor subtypes than to the rodent alpha2-adrenoceptors. We suggest a set of drugs that are useful for the delineation of the pig alpha2-adrenoceptor subtypes.