Management goals for type 1 Gaucher disease: An expert consensus document from the European working group on Gaucher disease.

Gaucher Disease type 1 (GD1) is a lysosomal disorder that affects many systems. Therapy improves the principal manifestations of the condition and, as a consequence, many patients show a modified phenotype which reflects manifestations of their disease that are refractory to treatment. More generally, it is increasingly recognised that information as to how a patient feels and functions [obtained by patient- reported outcome measurements (PROMs)] is critical to any comprehensive evaluation of treatment. A new set of management goals for GD1 in which both trends are reflected is needed. To this end, a modified Delphi procedure among 25 experts was performed. Based on a literature review and with input from patients, 65 potential goals were formulated as statements. Consensus was considered to be reached when ≥75% of the participants agreed to include that specific statement in the management goals. There was agreement on 42 statements. In addition to the traditional goals concerning haematological, visceral and bone manifestations, improvement in quality of life, fatigue and social participation, as well as early detection of long-term complications or associated diseases were included. When applying this set of goals in medical practice, the clinical status of the individual patient should be taken into account.

Granulocyte colony-stimulating factor-mobilized peripheral blood CD34+ cells from children contain the same levels of long-term culture-initiating cells producing the same numbers of colony-forming cells as those from adults, but display greater in vitro monocyte/macrophage potential.

Autologous peripheral blood progenitor cell (PBPC) transplantation is now commonly used in children. The ontogenic differences in haematopoiesis published in recent years suggest differences in the categories of mobilized PBPCs between children and adults. We investigated the frequency and distribution of mature progenitor cells (colony-forming cells, CFCs) and primitive progenitor cells [CD34+ CD38- and CD34+ Thy-1+ cells, long-term culture-initiating cells (LTC-ICs)] in children and adults mobilized using granulocyte colony-stimulating factor alone. We found similar proportions of granulocyte colony-forming units (CFU-G) and/or macrophage CFUs (CFU-M), mixed lineage CFUs (CFU-Mix) and megakarocyte CFUs (CFU-Mk), CD34+ CD38- and CD34+ Thy-1+ cells, and LTC-ICs (16.5 +/- 3.5 vs. 10.65 +/- 5 per 104 CD34+ cells), which produced the same number of CFCs (5 +/- 1 vs. 6 +/- 1 CFCs/LTC-ICs) in PB CD34+ cells from children and adults. However, we noted a higher proportion of erythroid blast-forming units (BFU-E) in PB CD34+ cells from adults (x 1.5, P = 0.003). Using cord blood as a third ageing point, we observed an inverse age-related propensity for commitment to the monocyte/macrophage lineage that was still found after normalizing the data per body weight and processed blood mass. This ontogeny-related programming was detected from the LTC-IC level, which produced 1.7 times more CFU-M in children than in adults (P = 0.048). These subtle differences in commitment between children and adults, shown here for the first time, are of interest for the in vitro manipulation of PBPCs and, in particular, for application in adoptive immunotherapy in children.

CFU-Mk content of immunoselected CD34+ peripheral blood progenitor cells, evaluated with an adapted serum-free methylcellulose assay, is predictive of platelet lineage reconstitution in children with solid tumors.

Immunoselected CD34+ peripheral blood progenitor cell (PBPC) transplantation is now frequently used to support autologous hematopoiesis after myeloablative therapy, its feasability having been proved by several groups. However, we and others observed delayed platelet recovery. We hypothesized that immunoselection processing might induce selective loss of megakaryocyte progenitors, or a decrease in their proliferation. We used a colony-forming units megakaryocyte (CFU-Mk) assay to evaluate these consequences and predict platelet recovery in patients. In CD34+ PBPCs from 10 children with solid tumors, we observed no selective loss in CFU-Mk numbers during immunoselection processing and no impairment of clonogenicity. The CFU-Mk yield (59.2 +/- 11.3%) was at least similar to the CD34+ yield (44.2 +/- 3.8%). We assessed the predictive value of CFU-Mk numbers infused for recovery of platelet lineage. We found an inverse correlation between the time taken to reach a platelet count greater than 50 x 10(9)/L and only the CFU-Mk dose (r = -0.71; p = 0.022) among the different type of progenitors, including colony-forming units granulocyte-macrophage (CFU-GM), burst-forming units erythrocyte (BFU-E) and colony-forming units-mixed (CFU-Mix). These findings suggest that CFU-Mk number could be used as sole predictive functional parameter for platelet reconstitution in children after immunoselection of CD34+ cells, in particular for low CD34+ cell dose, and thus as an indicator for initial quality of hematopoietic cells before in vitro expansion.

Rapid differentiation of a rare subset of adult human lin(-)CD34(-)CD38(-) cells stimulated by multiple growth factors in vitro.

Recently, several reports of lineage-negative (lin(-)) CD34(-) cells with in vivo hematopoietic activity have focused interest on the properties and growth factor response characteristics of these cells. We have now identified a combination of 5 growth factors that are necessary and sufficient to stimulate a marked mitogenic and differentiation response by a subset of human lin(-)CD34(-)CD38(-) cells present in normal adult human marrow and granulocyte colony-stimulating factor (G-CSF)-mobilized blood. Less than 0.1% of the cells in highly purified (including doubly sorted) lin(-)CD34(-)CD38(-) cells from these 2 sources formed colonies directly in semisolid medium or generated such cells after 6 weeks in long-term culture. Nevertheless, approximately 1% of the same lin(-)CD34(-)CD38(-) cells were able to proliferate rapidly in serum-free liquid suspension cultures containing human flt-3 ligand, Steel factor, thrombopoietin, interleukin-3 (IL-3), and hyper-IL-6 to produce a net 28- +/- 8-fold increase in total cells within 10 days. Of the cells present in these 10-day cultures, 5% +/- 2% were CD34(+) and 2.5% +/- 0.9% were erythroid, granulopoietic, megakaryocytopoietic, or multilineage colony-forming cells (CFC) (13 +/- 7 CFC per lin(-)CD34(-)CD38(-) pre-CFC). In contrast to lin(-)CD34(+)CD38(-) cells, this response of lin(-)CD34(-)CD38(-) cells required exposure to all of the 5 growth factors used. Up to 1.7 x 10(5) lin(-)CD34(-) adult marrow cells failed to engraft sublethally irradiated NOD/SCID-beta(2)M(-/-) mice. These studies demonstrate unique properties of a rare subset of lin(-)CD34(-)CD38(-) cells present in both adult human marrow and mobilized blood samples that allow their rapid proliferation and differentiation in vitro within an overall period of 3 to 4 weeks. The rapidity of this response challenges current concepts about the normal duration and coordinated control of these processes in adults.

Intracellular acidosis differentially regulates KV channels in coronary and pulmonary vascular muscle.

Decreases in intracellular pH (pHi) potently dilate coronary resistance arteries but constrict small pulmonary arteries. To define the ionic mechanisms of these responses, this study investigated whether acute decreases in pHi differentially regulate K+ currents in single vascular smooth muscle (VSM) cells isolated from rat coronary and pulmonary resistance arteries. In patch-clamp studies, whole cell K+ currents were elicited by 10-mV depolarizing steps between -60 and 0 mV in VSM cells obtained from 50- to 150-micrometers-OD arterial branches, and pHi was lowered by altering the NH4Cl gradient across the cell membrane. Progressively lowering pHi from calculated values of 7.0 to 6.7 and 6.4 increased the peak amplitude of K+ current in coronary VSM cells by 15 +/- 5 and 23 +/- 3% but reduced K+ current in pulmonary VSM cells by 18 +/- 3 and 21 +/- 3%, respectively. These changes were reversed by returning cells to the control pHi of 7.0 and were eliminated by dialyzing cells with pipette solution containing 50 mmol/l HEPES to buffer NH4Cl-induced changes in pHi. Pharmacological block of ATP-sensitive K+ channels and Ca2+-activated K+ channels by 1 micromol/l glibenclamide and 100 nmol/l iberiotoxin, respectively, did not prevent changes in K+ current levels induced by acidotic pHi. However, block of voltage-gated K+ channels by 3 mmol/l 4-aminopyridine abolished acidosis-induced changes in K+ current amplitudes in both VSM cell types. Interestingly, alpha-dendrotoxin (100 nmol/l), which blocks only select subtypes of voltage-gated K+ channels, abolished the acidosis-induced decrease in K+ current in pulmonary VSM cells but did not affect the acidosis-induced increase in K+ current observed in coronary VSM cells. These findings suggest that opposing, tissue-specific effects of pHi on distinct subtypes of voltage-gated K+ channels in coronary and pulmonary VSM membranes may differentially regulate vascular reactivity in these two circulations under conditions of acidotic stress.

Factors influencing the duration of breastfeeding in the Sudbury region.

OBJECTIVE: To determine the duration of breastfeeding in the Sudbury Region and to identify the reasons why mothers wean before the Canadian Paediatric Society's recommended six month period. METHODS: Questionnaires addressing factors that influence the duration of breastfeeding were mailed to 350 breastfeeding mothers at one/two weeks, three months and six months after their postpartum discharge from hospital. RESULTS: Forty percent of mothers breastfed for the recommended six month period. Reported factors positively influencing longer durations of breastfeeding were higher education, higher family income, parity, previous breastfeeding experience, decision to breastfeed before the child was born and late introduction to solids. Reasons for weaning included perceived insufficient milk supply, fatigue, breast problems and return to work. Mothers' top three choices of services were home visits, telephone hot line and television programs. CONCLUSIONS: The duration of breastfeeding in the Sudbury Region is lower than the provincial average. Several modifiable factors associated with duration of breastfeeding were identified.

Stress measurement in alumina scales on high temperature alloys using X-ray stress evaluation and laser Raman spectroscopy.

The effect of silicon and titanium on the spallation resistance of alumina scales grown on NiCrAlY-type alloys has been investigated using model alloys with different additions of Si or Ti. For this purpose cyclic oxidation experiments have been carried out at temperatures between 950 and 1100 degrees C. After various times stresses in selected Si-doped samples have been determined by X-ray stress evaluation (XSE) at ambient temperature. The compressive stresses in the scales have been found to increase with an increasing oxidation time tending to become constant for long times. The development of stress is affected by the presence of Si. Laser Raman spectroscopy (LRS) has been calibrated for strain measurement using XSE results. Then LRS has been applied for strain measurement at higher temperatures.

Implementation of a systematic health maintenance protocol in a private practice.

The impact of a policy to introduce a simple health maintenance protocol systematically to all patients belonging to a private practice is reported. Results after 18 months' experience in over 1400 patients indicate that (1) physician compliance was excellent (97% of eligible patients were included, and physician time taken to introduce the protocol at the index visit took less than 4 minutes), and (2) patient acceptance (which varied from procedure to procedure) was good to excellent (minimum acceptance: 77% for sigmoidoscopy; maximum acceptance: 97% for cholesterol screening). For patients seen once, acceptance rates for procedures were generally comparable to prior published performance rates for highly selected patient populations. Integration of a simple health maintenance protocol into routine office care of unselected primary care patients was feasible, effective, and acceptable to patients. Patient refusal was a minor barrier to performance of health maintenance.

The involvement of glutamine synthetase/glutamate synthase in ammonia assimilation by Aspergillus nidulans.

Wild-type Aspergillus nidulans grew equally well on NH4Cl, KNO3 or glutamine as the only nitrogen source. NADP+-dependent glutamate dehydrogenase (EC 1.4.1.4) and glutamine synthetase (GS; EC 6.3.1.2) activities varied with the type and concentration of nitrogen source supplied. Glutamate synthase (GOGAT) activity (EC 1.4.7.1) was detected but it was almost unaffected by the type and concentration of nitrogen source supplied. Ion exchange chromatography showed that the GOGAT activity was due to a distinct enzyme. Azaserine, an inhibitor of the GOGAT reaction, reduced the glutamate pool by 60%, indicating that GOGAT is involved in ammonia assimilation by metabolizing the glutamine formed by GS.

Ammonia Fixation via Glutamine Synthetase and Glutamate Synthase in the CAM Plant Cissus quadrangularis L.

Succulent stems of Cissus quadrangularis L. (Vitaceae) contain glutamine synthetase, glutamate synthase, and glutamate dehydrogenase. The CO(2) and water gas exchanges of detached internodes were typical for Crassulacean acid metabolism plants. During three physiological phases, e.g. in the dark, in the early illumination period after stomata closure, and during the late light phase with the stomata wide open, (15)NH(4)Cl was injected into the central pith of stem sections. The kinetics of (15)N labeling in glutamate and glutamine suggested that glutamine synthetase was involved in the initial ammonia fixation. In the presence of methionine sulfoximine, an inhibitor of glutamine synthetase, the incorporation of (15)N derived from (15)NH(4)Cl was almost completely inhibited. Injections of amido-(15)N glutamine demonstrated a potential for (15)N transfer from the amido group of glutamine into glutamate which was suppressed by the glutamate synthase inhibitor, azaserine. The evidence indicates that glutamine synthetase and glutamate synthase could assimilate ammonia and cycle nitrogen during all phases of Crassulacean acid metabolism.

Nitrogen Metabolism in Senescent Flag Leaves of Wheat (Triticum aestivum L.) in the Light.

Nitrogen metabolism was examined in senescent flag leaves of 90- to 93-day-old wheat (Triticum aestivum L. cv Yecora 70) plants. CO(2) assimilation and the levels of protein, chlorophyll, and nitrogen in the leaves decreased with age. Glutamine synthetase activity decreased to one-eighth of the level in young flag leaves. Detached leaves were incubated (with the cut base) in (15)N-labeled NH(3), glutamate, or glycine in the light (1.8 millieinstein per square meter per second) at 25 degrees C in an open gas exchange system under normal atmospheric conditions for up to 135 minutes. The (15)N-enrichment of various amino acids derived from these (15)N-substrates were examined. The amido-N of glutamine was the first (15)N-labeled product in leaves incubated with (15)NH(4)Cl whereas serine, closely followed by the amido- and amino-N of glutamine, were the most highly (15)N-labeled products during incubation with [(15)N]glycine. In contrast, aspartate and alanine were the first (15)N-labeled products when [(15)N] glutamate was used. These results indicate that NH(3) was assimilated via glutamine synthetase and glutamate synthase activities and the photorespiratory nitrogen cycle remained functional in these senescent wheat flag leaves. In contrast, an involvement of glutamate dehydrogenase in the assimilation of ammonia could not be detected in these tissues.

Effect of light intensity on ammonia assimilation in maize leaves.

The effect of light on the metabolism of ammonia was studied by subjecting detached maize leaves to 150 or 1350 µmol m(-2) s(-1) PAR during incubation with the leaf base in 2 mM (15)NH4Cl. After up to 60 min, leaves were extracted. Ammonia, glutamine, glycine, serine, alanine, and aspartate were separated by isothermal distillation and ion exchange chromatography. (15)N enrichments were analyzed by emission spectroscopy. The uptake of ammonium chloride did not influence CO2 assimilation (8.3 and 17.4 µmol m(-1) s(-1) at 150 and 1350 µmol m(-2) s(-1) PAR, respectively). Leaves kept at high light intensity contained more serine and less alanine than leaves from low light treatments. Within 1 h of incubation the enrichment of ammonia extracted from leaves rose to approximately 20% (15)N. In the high light regime the amino acids contained up to 15% (15)N, whereas in low light (15)N enrichments were small (up to 6%). The kinetics of (15)N incorporation indicated that NH3 was firstly assimilated into glutamine and then into glutamate. After 15 min (15)N was also found in glycine, serine and alanine. At high light intensity nearly half of the (15)N was incorporated in glycine. On the other hand, at low light intensity alanine was the predominant (15)N sink. It is concluded that light influences ammonia assimilation at the glutamine synthetase reaction.

(15)N and inhibitor studies on the photorespiratory nitrogen cycle in maize leaves.

A combination of inhibitor and (15)N studies were used to investigate the photorespiratory nitrogen cycle in maize, a C4 plant. Inhibitors used included isonicotinyl hydrazide which blocks the conversion of glycine to serine, methionine sulfoximine an inhibitor of GS and azaserine an inhibitor of GOGAT. Results from levels of ammonia and amino acids and the distribution of (15)N into NH3, serine, glutamine and glutamate indicated that the photorespiratory N-cycle occurs in this C4 plant, but the rate of flux through this pathway is low as compared with that in C3 plants.