Showing papers by "Montreal Children's Hospital published in 1991"

A rapid procedure for extracting genomic DNA from leukocytes

Abstract: 1. Collect 5 ml of blood in a vacutainer tube (Becton Dickinson) containing EDTA and mix. 2. Make volume up to 10 ml with solution 1 (10 mM Tris pH 7.6; 10 mM KC1; 10 mM MgCy. 3. Add 120 /il Nonidet P40 (BDH) to lyse the cells. Mix well by inverting several times. 4. Spin down the nuclear pellet at 2000 rpm for 10 mins. 5. Pour off the supernatant without dislodging the pellet. The pellets can be stored frozen. 6. Gently resuspend pellet well in 800 /il of solution 2 (10 mM Tris pH 7.6; 10 mM KC1; 10 mM MgCl2; 0.5 M NaCl; 0.5% SDS; 2 mM EDTA). Solution 2 will lyse the nuclei so be careful not to shear the DNA. Transfer to a 1.5 ml microcentrifuge tube. 7. Add 400 /il of distilled phenol (saturated with 1 M Tris pH 8.0) and mix well. 8. Microfuge for 1 min at 12000 rpm. Transfer upper phase to a clean microfuge tube. Do not worry about transferring a little of the interface. 9. Add 200 /tl of phenol and 200 /il of chloroform :isoamyl alcohol (24:1). Mix well by inverting. 10. Spin for 1 min at 12000 rpm. Transfer upper phase to a clean microfuge tube. 11. Add 700 /tl of chloroform:isoamyl alcohol and extract as above. 12. Transfer upper aqueous phase to a small clean container. Avoid removing the interface. Add 2 volumes of ice cold ethanol and mix to precipitate the DNA*. 13. With the sealed tip of a pasture pipette, transfer the DNA fibres to a microcentrifuge tube containing 1 ml of 70% ethanol. Mix well to wash the DNA. 14. Spin for 5 mins at full speed. Discard the ethanol and dry pellet in a speed vac. Resuspend DNA in sterile water at 65°C. Do not over-dry genomic DNA or it will be difficult to resuspend. SIZE A / /Kb Hindll l

Lower incidence of meningeal leukemia when prednisone is replaced by dexamethasone in the treatment of acute lymphocytic leukemia.

Abstract: In 1971, Cancer and Leukemia Group B (CALGB) mounted a study of acute lymphocytic leukemia (ALL) that compared the effects of the two steroid hormones dexamethasone and prednisone. Six-hundred-forty-six children and adolescents with ALL were randomized to receive either prednisone or dexamethasone as part of their remission induction therapy. The 493 evaluable patients who achieved complete remission received the same steroid as pulses throughout remission. Specific central nervous system (CNS) therapy was randomized to either six injections of intrathecal methotrexate (IT MTX) alone or to six injections of IT MTX with cranial radiation (2,400 cGy). Both cranial radiation and dexamethasone offered increased protection against CNS relapse as the first site of failure over IT MTX alone. There were 30 CNS relapses among 238 patients (12.6%) receiving cranial radiation plus IT MTX, whereas there were 70 CNS relapses among 225 (P less than 0.001) (22.5%) in those who received IT MTX alone. Similarly, there were 33 CNS relapses among 231 (14.3%) children treated with dexamethasone, whereas there were 67 CNS relapses among 262 (25.6%) treated with prednisone (P = 0.017). Both steroids appeared equal in protecting the bone marrow. Recent national studies have shown significant improvements in preventing CNS relapse over the results in the present report. However, this finding warrants further investigation and, with further documentation, could lead to the substitution of prednisone by dexamethasone to aid further in preventing CNS relapse. This may be particularly important in patients at higher risk for CNS relapse.

Sexual dimorphism of somatostatin and growth hormone-releasing factor signaling in the control of pulsatile growth hormone secretion in the rat.

Abstract: A striking sexual dimorphism exists in the pattern of GH secretion and rate of somatic growth; however, the mechanism(s) mediating this sex difference is unknown. To elucidate the physiological roles of the hypothalamic neuropeptides, somatostatin (SRIF) and GRF, and their interrelation, in generating the sexually dimorphic GH secretory pattern we examined: 1) GH responsiveness to exogenous GRF and 2) the effects of immunoneutralization of endogenous SRIF and GRF on GH secretory dynamics, in free-moving male and female rats. In males, the GH response to 1 microgram rat(r)GRF(1-29)NH2 iv was significantly greater at peak compared to trough times of GH secretion (925.2 +/- 250.8 vs. 95.6 +/- 27.8 ng/ml; P less than 0.02), the latter known to be due to antagonization by the cyclic increased release of endogenous SRIF. In contrast, females failed to exhibit a time-dependent difference in GH responsiveness to GRF. Passive immunization with a specific antiserum to SRIF in males resulted in significant elevation of GH nadir levels but had no effect on GH peak amplitude. In contrast, immunoneutralization of endogenous SRIF in females caused a marked augmentation of plasma GH levels at all time points; there was a significant increase in GH peak amplitude (171.3 +/- 39.9 vs. 67.5 +/- 11.3 ng/ml; P less than 0.05), GH nadir (18.3 +/- 2.7 vs. 5.8 +/- 1.1 ng/ml; P less than 0.01) and mean 6-h plasma GH level (78.7 +/- 4.1 vs. 33.1 +/- 5.8 ng/ml; P less than 0.001), compared to normal sheep serum-treated controls. These results indicate that the pattern of hypothalamic SRIF secretion in females does not follow the male-like ultradian rhythm. Passive immunization with a specific antiserum to GRF obliterated spontaneous GH pulses in both sexes. Moreover, in females, anti-GRF serum attenuated GH nadir levels (4.3 +/- 1.7 vs. 21.4 +/- 3.5 ng/ml; P less than 0.01) indicating a physiological role for GRF in maintaining the elevated basal GH level of females, in addition to its important role in generating the episodic GH pulses. Taken together, these findings provide support for the hypothesis that, in female rats, the pattern of hypothalamic SRIF secretion into hypophyseal portal blood is continuous, rather than cyclical, as in the male; whereas in the case of GRF secretion, in addition to steady-state release which occurs at a higher level in females than males, there is also episodic GRF bursting which does not follow a specific rhythm, as in the male.(ABSTRACT TRUNCATED AT 400 WORDS)

Differences in the effects in the newborn piglet of various nonsteroidal antiinflammatory drugs on cerebral blood flow but not on cerebrovascular prostaglandins.

Abstract: To characterize the role of prostaglandins (PG) in the regulation of basal cerebral blood flow (CBF) in the newborn, we determined the effects of four nonsteroidal antiinflammatory drugs, indomethacin (3 mg/kg, n = 8 and 10 mg/kg, n = 5), aspirin (65 mg/kg, n = 6), ibuprofen (30 mg/kg, n = 8), and naproxen (15 mg/kg, n = 6), on CBF, cerebral metabolism, and cerebrovascular PG in conscious 1- to 3-d-old piglets. Drugs and vehicle (n = 8) were injected i.v., and measurements were made 5 min before and 20 and 60 min after injections. Neither the vehicle nor any of the nonsteroidal antiinflammatory drugs exerted significant effects on mean arterial blood pressure and on blood gases and pH. All four drugs, with the exception of indomethacin at the lower dose (3 mg/kg), decreased PG to nearly undetectable levels within 20 min; the low dose of indomethacin caused a small decrease (18-32%) in PG at 60 min. However, the effects of these agents on CBF were diverse. CBF increased after the administration of aspirin, decreased to almost the same extent after both low and high doses of indomethacin, and did not change after the administration of ibuprofen and naproxen. Cerebral metabolic rate for oxygen was increased by aspirin but was unaltered by the other drugs. The data suggest that PG may not play a critical role in the regulation of basal CBF in the newborn animal and that certain nonsteroidal antiinflammatory drugs may have additional actions unrelated to the inhibition of PG synthesis.

Study strategies and story recall in attention deficit disorder and reading disability

Abstract: The role of metacognition and executive processes in mediating use of study skills was examined in groups of attention deficit disorder with hyperactivity (ADD-H), normal, and non-ADD-H reading-disabled (RD) boys, matched on age and verbal IQ. On a story recall task, ADD-H boys did not differ from normals in their immediate gist recall of a story or in their recall following a study period. RD boys demonstrated inferior recall in both conditions. Study skills of the ADD-H boys were poorer than those of normal boys on all measures. They spent less time studying, expended less effort, and employed more superficial strategies. However, their poor strategies did not appear to reflect a lack of metacognitive awareness. Results are discussed in terms of the impact of motivational variables in modulating strategy use in ADD-H boys and the impact of verbal processing problems in reading disabilities. Implications for treatment and the relationship between ADD-H and RD are discussed.

Sequence of DNA flanking the exons of the HEXA gene, and identification of mutations in Tay-Sachs disease.

Abstract: The rapid identification of mutations causing Tay-Sachs disease requires the capacity to readily screen the regions of the HEXA gene most likely to be affected by mutation. We have sequenced the portions of the introns flanking each of the 14 HEXA exons in order to specify oligonucleotide primers for the PCR-dependent amplification of each exon and splice-junction sequence. The amplified products were analyzed, by electrophoresis in nondenaturing polyacrylamide gels, for the presence of either heteroduplexes, derived from the annealing of normal and mutant DNA strands, or single-strand conformational polymorphisms (SSCP), derived from the renaturation of single-stranded DNA. Five novel mutations from Tay-Sachs disease patients were detected: a 5-bp deletion of TCTCC in IVS-9; a 2-bp deletion of TG in exon 5; G78 to A, giving a stop codon in exon 1; G533 to T in exon 5, producing the third amino acid substitution detected at this site; and G to C at position 1 of IVS-2, expected to produce abnormal splicing. In addition, two mutations, (G1496 to A in exon 13 and a 4-bp insertion in exon 11) that have previously been reported were identified.

Resilience and vulnerability in long term outcome of attention deficit hyperactive disorder.

Abstract: Follow-up studies of children with attention deficit hyperactive disorder show that they continue to have problems with restlessness, over-activity, impulsive behaviour and inattention, often resul...

Measurement of respiratory system resistance by forced oscillation in normal children: a comparison with spirometric values

Abstract: In 377 children, the commercially available Siregnost FD-5 was used to measure respiratory system resistance (Rrs) by forced oscillation at 10 Hz. The children were between 3 and 18 years of age and, by a detailed questionnaire and conventional pulmonary function testing in 335, they were shown to be representative of the normal pediatric population. There was a linear relationship between Rrs and height (Rrs = 13.9-0.064 x ht (cm), r = -0.87). Children less than 6 years of age had no trouble with using the forced oscillation technique. The smoking of tobacco in the house, the presence of carpets in the child's bedroom, or an atopic family history, alone or in combination, had no influence on Rrs or on any spirometric measure. Forced oscillation is useful in children too young to be able to cooperate with conventional pulmonary function testing.

Ibuprofen enhances retinal and choroidal blood flow autoregulation in newborn piglets.

Abstract: The role of prostanoids in setting the range of autoregulation of retinal blood flow (RBF) and choroidal blood flow (ChBF) in the newborn was assessed. The RBF, ChBF, and arterial and cerebral sinus concentrations of PGE, PGF2 alpha, 6-keto-PGF1 alpha and TXB2 were measured over a wide range of mean systemic blood pressure (blood pressure (BP): 17-117 mm Hg) in newborn piglets treated with ibuprofen (30 mg/kg iv) or its vehicle (n = 8, in each group). Hypertension and hypotension were induced 80 min apart on each animal, by inflating balloon-tipped catheters placed at the aortic isthmus and root, respectively. Blood flow and prostanoid concentrations were measured 20 min before (basal) and during the induced changes in BP. In vehicle-treated piglets, RBF did not change with BP between 50 and 90 mm Hg (r = 0.33, P = 0.27), but changed as a function of BP beyond this range (tau = 0.52, P less than 0.01); ChBF increased with BP throughout the range studied (17-117 mm Hg; tau = 0.89, P less than 0.001). The relationship between O2 delivery to the retina and choroid and BP (tau greater than 0.43, P less than 0.01) was similar to that seen between RBF and ChBF with BP. The concentration of all prostanoids increased when BP was reduced to less than 50 mm Hg. When BP was raised to more than 90 mm Hg, prostaglandin concentrations increased, and those of TXB2 did not change. Ibuprofen treatment reduced the basal concentrations of all prostanoids to nearly undetectable levels and prevented their changes during hypotension and hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

Severe Lactic Acidosis Related to Acute Thiamine Deficiency

Abstract: The authors report a case of severe lactic acidosis in a 3-year-old boy, after 20 days of total parenteral nutrition without vitamin supplementation. This child with acute lymphoblastic leukemia underwent a period of severe refractory lactic acidosis (pH between 6.81 and 7.00 and a serum lactate level up to 38 mmol/liter) leading to cardiac arrest. After the initial resuscitation and the subsequent treatment of shock and vitamin K deficiency, acute peritoneal dialysis was instituted to correct the severe lactic acidosis. Initial low plasma thiamine levels confirmed the diagnosis of thiamine deficiency. An associated transient pancreatic dysfunction was also noted. The patient's overall course with thiamine replacement therapy led to a complete recovery within 5 days and no sequelae were noted after 12 months.

Limb reconstruction with vascularized fibular grafts after bone tumor resection.

Abstract: Limb-salvage operations are being used with increasing frequency for patients with malignant bone tumors. For children, when a biologic reconstruction is desired, the choice is often between conventional and vascularized fibular grafts. An experimental study was performed in dogs to compare the two types of fibular grafts for bridging segmental defects in the radius and ulna. Twenty-six adult dogs were divided into two groups and studied at intervals of two, three, four, six, and 12 months after transplantation. The conventional grafts healed by creeping substitution i.e., they were first partially resorbed before new bone was laid down. In contrast, the vascularized fibulae maintained their normal structure and hypertrophied by subperiosteal new bone formation. The conventional fibulae eventually hypertrophied but much later than the vascularized grafts. The vascularized grafts were stronger at four and six months. Between six and 12 months, both grafts remodeled to resemble the size and shape of the forearm bones they were replacing. These experimental results have influenced the treatment of patients. Vascularized fibular grafts are ideal for diaphyseal defects greater than 10 cm long, especially in very young children, a poorly vascularized bed, or when bone healing is delayed by chemotherapeutic agents. To maximize hypertrophy, an external fixator is used to immobilize the graft rather than a plate, which acts as a stress shield.

Corticostatic peptides cause nifedipine-sensitive volume reduction in jejunal villus enterocytes.

Abstract: We studied cell-volume changes caused by adding corticostatin (CS) or defensin-like peptides to villus enterocytes isolated in suspension from guinea pig jejunum. Guinea pig CS (10(-9) M) added to villus cells in Na(+)-containing medium reduced volume, but immediate cell swelling was caused by 10(-6) M guinea pig CS. In Na(+)-free N-methyl-D-glucamine-containing medium 10(-9) M guinea pig CS accelerated the initial rate of shrinkage compared with cells in N-methyl-D-glucamine-containing medium alone as well as causing greater cell shrinkage. Guinea pig CS-stimulated cell shrinkage was prevented by a Ca2(+)-channel blocker--5 microM nifedipine, by chelation of extracellular Ca2+ with 100 microM EGTA, or by omega-conotoxin (10(-9) M). The Ca2+ ionophore A23187 (2.5 microM) reduced volume when added to villus cells in N-methyl-D-glucamine-containing medium; this action was prevented by EGTA, or quinine--an inhibitor of K+ conductance, or 9-anthracenecarboxylic acid--a Cl- channel blocker, suggesting that the volume reduction occurred because K+ and Cl- conductances were activated. Guinea pig CS-stimulated volume reduction was also prevented by 100 microM quinine or 9-anthracenecarboxylic acid. We conclude that jejunal villus enterocytes possess a Ca2(+)-activated Cl- conductance and a K+ conductance that need not be stretch-activated. Corticostatic peptides cause volume reduction in villus cells by activating L-type Ca2+ channels; other defensin-like peptides were without effect.

Possible common pathogenetic mechanisms for Poland sequence and Adams-Oliver syndrome.

Abstract: We report on 2 families having members affected with the Poland sequence and Adams-Oliver syndrome. In the first family, a 5-month-old boy presented with Adams-Oliver syndrome; his mother had Poland sequence. In the second family, a 12-year-old boy and his mother presented with findings suggestive of Adams-Oliver syndrome and Poland sequence. This suggests that the same genetic predisposition may result in either or both conditions.

Volume regulation initiated by Na(+)-nutrient cotransport in isolated mammalian villus enterocytes.

Abstract: We assessed ion transport during regulatory volume decrease (RVD) in jejunal villus enterocytes, isolated in suspension from guinea pig jejunum and swollen by exposure to L-alanine (L-Ala) or D-glucose (D-Glc) in the presence of Na+. Cell volume was measured electronically. Relative volume of cells (rel vol: cell vol/isotonic vol) within 1 min of L-Ala (20 mM) addition increased (1.10 +/- 0.03, P less than 0.005), but by 5 min there was no difference between cells in L-Ala or 20 mM D-Ala (0.95 +/- 0.02). Cell shrinkage after maximal swelling was greater with L-Ala than with D-Ala (14 +/- 4 vs. 2 +/- 1%, P less than 0.01). Initial swelling generated by L-Ala required extracellular Na+ (P less than 0.02). Volume increased 30 s after D-Glc (20 mM), and cells were larger than cells treated with L-Glc (1.04 +/- 0.01 vs. 0.95 +/- 0.01, P less than 0.001); subsequent cell shrinkage was complete in 2 min (8 +/- 2%, P less than 0.05). Swelling generated by methyl alpha-D-glucoside was prevented by 0.1 mM phloridzin (P less than 0.05). RVD after D-Glc swelling was prevented by inhibitors of K+ channels, 5 mM Ba2+ (P less than 0.001), 100 microM quinine (P less than 0.005), or 25 mM TEA (P less than 0.02), but the same inhibitors completely prevented L-Ala swelling. All inhibitors had no effect on L-Ala uptake into brush-border membrane vesicles in presence of Na+ gradient.(ABSTRACT TRUNCATED AT 250 WORDS)

Echogenic renal pyramids in children.

Abstract: A retrospective study of 68 children with increased medullary echogenicity on renal ultrasound (US) examination showed nephrocalcinosis to be present in 42 patients. The cause was believed to be iatrogenic in 30 and noniatrogenic in 12. Furosemide therapy was responsible for 11 of the cases of iatrogenic nephrocalcinosis and vitamin D therapy for the remaining iatrogenic cases. Noniatrogenic nephrocalcinosis was seen with hypercalcemia, hypercalciuria, renal tubular acidosis and dystrophic calcification following renal tubular necrosis. In 26 patients, medullary deposits of urates or proteins, medullary fibrosis, or vascular congestion (due to a variety of diseases) appeared to account for the finding. These possibilities should be added to the differential diagnosis of hyperechoic renal pyramids when nephrocalcinosis is unlikely.

Metabolic effects of growth factors and polycyclic aromatic hydrocarbons on cultured human placental cells of early and late gestation

Abstract: The metabolic effects of epidermal growth factor (EGF), insulin, insulin-like growth factor-I (IGF-I), and IGFII were determined on human placental cells in monolayer culture obtained from early gestation (<20 weeks) and late gestation (38–42 weeks). Parameters studied were uptake of aminoisobutyric acid (AIB), uptake of 3-O-methylglucose and [3H]thymidine incorporation into cell protein. Since benzo[α] pyrene (BP) inhibits EGF binding and autophosphorylation in cultured human placental cells, particularly in early gestation, we also studied the effect of benzo[α]pyrene and other polycyclic aromatic hydrocarbons (PAHs) on EGF-mediated AIB uptake. The metabolic effects of EGF, insulin, and the IGFs in cultured human placental cells varied with gestational age and the growth factor studied. All three classes of growth factors stimulated AIB uptake in both early and late gestation at concentrations from 10–100 μg/L, well within a physiological range. However, insulin stimulation of AIB uptake was maximal at ...

Deficiency of complex III of the mitochondrial respiratory chain in a patient with facioscapulohumeral disease.

Abstract: Facioscapulohumeral disease (FSHD), an inherited neuromuscular disorder, is characterized by progressive wasting of specific muscle groups, particularly the proximal musculature of the upper limbs; the primary defect in this disorder is unknown. We studied a patient with FSHD to determine whether the mitochondrial respiratory chain was functionally abnormal. Muscle biopsy revealed fiber atrophy with patchy staining for oxidative enzymes. Electron microscopy of a liver section showed many enlarged mitochondria with paracrystalline inclusions. Decreased oxidation of the respiratory substrates-alanine and succinate-in skin fibroblasts suggested a deficiency of complex III of the electron-transport chain; cytochrome c oxidase activity (complex IV) was in the normal range. Biochemical analysis of liver supported the fibroblast data, since succinate oxidase activity (electron-transport activity through complexes II-IV) was reduced, whereas complex IV activity was normal. Furthermore, analysis of the cytochrome spectrum in liver revealed typical peaks for cytochromes cc1 and aa3, whereas cytochrome b (a component of complex III) was undetectable. Southern blot analysis of fibroblast mtDNA revealed no major deletions or rearrangements. Our study provides the first documentation of a specific enzyme-complex deficiency associated with FSHD.

Generalized enchondromatosis in a boy with only platyspondyly in the father

Abstract: We report on a boy with platyspondyly and metaphyseal manifestations of enchondromatosis with severe involvement of hands and feet, compatible with generalized enchondromatosis (enchondromatosis Spranger type VI). His father has only moderate platyspondyly. The latter is either coincidental or a milder, more localized expression of the disease. Father and son have consanguineous parents suggesting autosomal recessive inheritance of the trait. However, autosomal dominant inheritance with variable expressivity is also possible.