Single nucleotide polymorphism in IL1B is associated with infection risk in paediatric acute myeloid leukaemia.

We evaluated single nucleotide polymorphisms (SNPs) associated with infection risk in children with newly diagnosed acute myeloid leukaemia (AML). We conducted a multicentre, prospective cohort study that included children aged ≤18 years with de novo AML. DNA was isolated from blood lymphocytes or buccal swabs, and candidate gene SNP analysis was conducted. Primary outcome was the occurrence of microbiologically documented sterile site infection during chemotherapy. Secondary outcomes were Gram-positive and -negative infections, viridans group streptococcal infection and proven/probable invasive fungal infection. Interpretation was guided by consistency in risk alleles and microbiologic agent with previous literature. Over the study period 254 children and adolescents with AML were enrolled. Overall, 190 (74.8%) had at least one sterile site microbiologically documented infection. Among the 172 with inferred European ancestry and DNA available, nine significant associations were observed; two were consistent with previous literature. Allele A at IL1B (rs16944) was associated with decreased microbiologically documented infection, and allele G at IL10 (rs1800896) was associated with increased risk of Gram-positive infection. We identified SNPs associated with infection risk in paediatric AML. Genotype may provide insight into mechanisms of infection risk that could be used for supportive-care novel treatments.

Invasive fungal infections in paediatric acute myeloid leukaemia.

Invasive fungal infections (IFIs) are a major cause of morbidity and mortality in paediatric acute myeloid leukaemia (AML). This study describes risk factors for IFI and IFI-related sepsis in this population. We conducted a population-based, retrospective cohort study of children with AML in Canada. IFIs during chemotherapy and prior to haematopoietic stem cell transplantation, relapse, persistent disease or death were identified. Risk factors for proven or probable IFI were examined. Among courses complicated by IFI, risk factors for sepsis were also evaluated. There were 341 children with AML included of which 41 (12.0%) experienced 46 different episodes of IFI. Candida species accounted for 23 (50.0%) of IFIs and Aspergillus spp. accounted for 14 (30.4%). Days of broad-spectrum antibiotics, days of corticosteroids and neutropenia at start of the course were independently associated with IFI. Only days of fever were independently associated with IFI-related sepsis. Invasive fungal infections occurred in 12.0% of paediatric AML patients. Risk factors for IFI and IFI-related sepsis were identified. This knowledge may help to consider targeted strategies.

Clotting factor concentrates given to prevent bleeding and bleeding-related complications in people with hemophilia A or B.

BACKGROUND: People with severe hemophilia A or B, X-linked bleeding disorders due to decreased blood levels of coagulants, suffer recurrent bleeding into joints and soft tissues. Before clotting factor concentrates were available, most people with severe hemophilia developed crippling musculoskeletal deformities. Clotting factor concentrate prophylaxis aims to preserve joint function by converting severe hemophilia (factor VIII or IX less than 1%) into a clinically milder form of the disease. Prophylaxis has long been used in Sweden, but not universally adopted because of medical, psychosocial, and cost controversies. Use of clotting factor concentrates is the single largest predictor of cost in treating hemophilia. OBJECTIVES: To determine the effectiveness of clotting factor concentrate prophylaxis in the management of people with hemophilia A or B. SEARCH STRATEGY: We searched the Cystic Fibrosis and Genetic Disorders Group's Trials Register comprising references from comprehensive electronic database searches and handsearches of journals and abstract books. Reference lists of relevant articles were reviewed. Most recent search: November 2005. SELECTION CRITERIA: Randomized controlled trials (RCTs) evaluating people with severe hemophilia A or B, receiving prophylactic clotting factor concentrates. DATA COLLECTION AND ANALYSIS: Two authors independently reviewed studies for eligibility, assessed methodological quality and extracted data. MAIN RESULTS: Twenty-nine studies were identified; four studies (including 37 participants) were eligible for inclusion. Three studies evaluated hemophilia A; one showed a decrease in frequency of joint bleeds with prophylaxis compared to placebo (non-physiological dose), with a rate difference (RD) -10.80 (95% confidence interval (CI) -16.33 to -5.27) bleeds per year. The remaining two studies evaluating hemophilia A compared two prophylaxis regimens, one study showed no difference in joint bleed frequency, RD -5.04 (95%CI -17.02 to 6.94) bleeds per year and another failed to demonstrate an advantage of factor VIII dosing based on individual pharmacokinetic data over the standard prophylaxis regimen with RD -0.14 (95% CI -1.34 to 1.05) bleeds per year. The fourth study evaluated hemophilia B and showed fewer joint bleeds with weekly (15 IU/kg) versus bi-weekly (7.5 IU/kg) prophylaxis, RD -3.30 (95% CI -5.50 to - 1.10) bleeds per year. AUTHORS' CONCLUSIONS: There is insufficient evidence from randomised controlled trials to determine whether prophylactic clotting factor concentrates decrease bleeding and bleeding-related complications in hemophilia A or B, compared to placebo, on-demand treatment, or prophylaxis based on pharmacokinetic data from individuals. Well-designed RCTs are needed to assess the effectiveness of prophylactic clotting factor concentrates. Two clinical trials are ongoing.

Clotting factor concentrates given to prevent bleeding and bleeding-related complications in people with hemophilia A or B.

BACKGROUND: People with severe hemophilia A or B, X-linked bleeding disorders due to decreased blood levels of coagulants, suffer recurrent bleeding into joints and soft tissues. Before clotting factor concentrates were available, most people with severe hemophilia developed crippling musculoskeletal deformities. Clotting factor concentrate prophylaxis aims to preserve joint function by converting severe hemophilia (factor VIII or IX less than 1%) into a clinically milder form of the disease. Prophylaxis has long been used in Sweden, but not universally adopted because of medical, psychosocial, and cost controversies. Use of clotting factor concentrates is the single largest predictor of cost in treating hemophilia. OBJECTIVES: To determine the effectiveness of clotting factor concentrate prophylaxis in the management of people with hemophilia A or B. SEARCH STRATEGY: We searched the Cystic Fibrosis and Genetic Disorders Group's Trials Register comprising references from comprehensive electronic database searches and handsearches of journals and abstract books. Reference lists of relevant articles were reviewed. Most recent search: January 2002. SELECTION CRITERIA: Randomized controlled trials (RCTs) evaluating people with severe hemophilia A or B, receiving prophylactic clotting factor concentrates. DATA COLLECTION AND ANALYSIS: Two reviewers independently reviewed studies for eligibility, assessed methodological quality and extracted data. MAIN RESULTS: Twenty-nine studies were identified, of which four (including 37 participants) were eligible for inclusion. Three studies evaluated hemophilia A; one showed a decrease in frequency of joint bleeds with prophylaxis compared to placebo (non-physiological dose), with a rate difference (RD) -10.80 (95% confidence interval (CI) -16.33 to -5.27) bleeds per year. The remaining two studies evaluating hemophilia A compared two prophylaxis regimens, one study showed no difference in joint bleed frequency, RD -5.04 (95%CI -17.02 to 6.94) bleeds per year and another failed to demonstrate an advantage of factor VIII dosing based on individual pharmacokinetic data over the standard prophylaxis regimen with RD -0.14 (95% CI -1.34 to 1.05) bleeds per year. The fourth study evaluated hemophilia B and showed fewer joint bleeds with weekly (15 IU/kg) versus bi-weekly (7.5 IU/kg) prophylaxis, RD -3.30 (95% CI -5.50 to - 1.10) bleeds per year. AUTHORS' CONCLUSIONS: There is insufficient evidence to determine whether prophylactic clotting factor concentrates decrease bleeding and bleeding-related complications in hemophilia A or B, compared to placebo, on-demand treatment, or prophylaxis based on pharmacokinetic data from individuals. Well-designed RCTs are needed to assess the effectiveness of prophylactic clotting factor concentrates. Two clinical trials are ongoing.

Evidence-based treatment of haemophilia.

Haemophilia care is effective but therapy is expensive. Despite concentrates being available in some areas for more than 30 years, many scientific questions concerning the management of haemophilia, such as what doses and duration of factor are required for different bleeding episodes or for surgical procedures, remain inadequately answered. Modern rigorous methods of assessment of the evidence both assist in determining what facts are available, how reliable the evidence is, and also help to define what studies need to be done. Cochrane methodology can be applied to haemophilia, and an assessment of prophylaxis provides an example. International collaborative studies would be valuable and require enthusiastic participants.

Cloning of a palmitoyl-acyl carrier protein thioesterase from oil palm.

A palmitoyl-acyl carrier protein (ACP) thioesterase cDNA clone was isolated from an oil palm cDNA library. The cDNA was expressed in Escherichia coli as a glutathione S-transferase fusion protein and a crude bacterial extract was assayed for acyl-CoA-hydrolysing activity. The recombinant enzyme was able to hydrolyse medium- and long-chain acyl-CoAs. Northern-blot analysis showed a high level of gene expression in leaf, flower and 15-, 17- and 18-week mesocarp tissues. Low-level gene expression was detected in germinated seedlings and 8- and 12-week mesocarp tissues, but no transcript was detected in any kernel tissues. Southern-blot analysis indicated the presence of a single gene and we have also isolated a genomic clone using the cDNA as a probe. Two genomic fragments were subcloned and a 7 kb contiguous stretch of the oil palm genome was sequenced. Comparison of this sequence with the cDNA sequence identified a putative 93 amino acid transit peptide, most of which is missing from the cDNA. The coding region of the gene consisted of seven exons and six introns.

Partial purification and photoaffinity labelling of sunflower acyl-CoA:lysophosphatidylcholine acyltransferase.

Previous attempts to purify acyl-CoA:1-acyl-lysophosphatidylcholine acyltransferase (EC 2.3.1.23) have been frustrated by difficulties in solubilizing the enzyme without inactivation. Microsomal preparations, from the developing cotyledons of sunflower, in high concentrations of urea retain activity. Gel-filtration liquid chromatography followed by trypsin treatment (minus urea) resulted in the removal of many contaminating proteins without loss of enzyme activity. SDS/PAGE showed the presence of two major peptides with apparent molecular masses of 52 and 59 kDa. These polypeptides cross-reacted with the radiolabelled photoreactive substrate 1-azido-oleoyl-sn-lysophosphatidyl-[N-methyl-(3)H]choline.

Oil biosynthesis in microsomal membrane preparations from Mortierella alpina.

Microsomal membrane preparations from Mortierella alpina catalysed the conversion of sn-glycerol 3-phosphate and [(14)C]oleoyl-CoA to radioactive phosphatidic acid, diacylglycerol and triacylglycerol. Experiments with lysophosphatidic acid and [(14)C]oleoyl-CoA gave a similar pattern of radioactivity in the complex lipids. The specific activity of lysophosphatidate acyltransferase was almost eight times greater than sn-glycerol-3-phosphate acyltransferase, indicating that the first acylation step was limiting in oil assembly in the microsomal membranes. Little conversion of radioactive oleate into phosphatidylcholine occurred, suggesting that triacylglycerol assembly and its relationship to phosphatidylcholine metabolism differed to that found in oilseeds.

Does triacylglycerol biosynthesis require diacylglycerol acyltransferase (DAGAT)?

Microsomal membrane preparations from the developing seeds of sunflower (Helianthus annuus L.) catalyse the conversion of sn-glycerol-3-phosphate and acyl-CoA to triacylglycerol via phosphatidic acid and diacylglycerol. The formation of diacylglycerol from phosphatidic acid was Mg2+ dependent and in the presence of EDTA phosphatidic acid accumulated. This property was used to generate large quantities of endogenous radioactive phosphatidic acid in the membranes. On addition of Mg2+ the phosphatidic acid was used in triacylglycerol formation. Acyl-CoA had little effect on the label which accumulated in triacylglycerol from phosphatidic acid. Diacylglycerol acyltransferase, therefore, may not play a major role in oil formation as originally envisaged and other enzymes, including diacylglycerol:diacylglycerol transacylase [Stobart, Mancha, Lenman, Dahlqvist and Stymne (1997) Planta 203, 58-66] may have important biosynthetic functions.

Sesamin inhibits lysophosphatidylcholine acyltransferase in Mortierella alpina.

The filamentous fungus, Mortierella alpina, accumulates complex lipids relatively rich in arachidonic acid (C(20:4) Delta(5,8,11,14)). The lignan, sesamin, has been used to reduce arachidonic acid production by specifically inhibiting Delta(5)-desaturation [Shimizu, Akimoto, Shinmen, Kawashima, Sugano and Yamada (1991) Lipids 26, 512-516]. Microsomal membrane preparations from M. alpina exhibit acyl-CoA:1-acyl lysophosphatidylcholine acyltransferase (LPCAT) activity. LPCAT is an enzyme involved in channelling fatty acid substrates to phosphatidylcholine for subsequent desaturation. Sesamin was found to inhibit this enzyme as measured in both spectrophotometric and radioactive assays. The inhibitory effect of sesamin on LPCAT was only evident in species of Mortierella and could not be demonstrated in other organisms.

Allogeneic bone marrow transplantation for an adolescent with polycythemia vera.

Polycythemia vera is a rare clonal disorder of the bone marrow haematopoietic stem cell. We present the case of an adolescent female with polycythemia vera, who was treated with a curative HLA-matched allogeneic bone marrow transplant.

Biosynthesis of vernoleate (cis-12-epoxyoctadeca-cis-9-enoate) in microsomal preparations from developing endosperm of Euphorbia lagascae.

Epoxidated fatty acids are the major constituents of the triacylglycerols in a few plant species. We have investigated the biosynthesis of vernolic acid (cis-12-epoxyoctadeca-cis-9-enoic acid) in the seed oil of Euphorbia lagascae. Microsomes were isolated from developing endosperm. The membrane lipids were labeled in situ with [14C]oleate or [14C]linoleate, which mainly were recovered in phosphatidylcholine (PC), and the metabolization of the radioactive fatty acids was followed in incubations with or without NADPH. In the presence of NADPH, [14C]vernoleate was formed. After short incubations, most of the vernolic acid was found in PC, but with increasing incubation times, the free acid dominated. The synthesis of vernoleate was inhibited by carbon monoxide, but not by cyanide. The presence of anticytochrome b5 antibodies inhibited both the desaturation of [14C]oleate to [14C]linoleate and the epoxidation of [14C]linoleate to [14C]vernoleate. Free linoleic acid did not serve as substrate for epoxidation. The results indicate that, in the endosperm of E. lagascae, vernoleate is synthesized on PC from linoleate, and that the epoxidation is catalyzed by a cytochrome P450 and involves cytochrome b5.

Evidence for cytochrome b5 as an electron donor in ricinoleic acid biosynthesis in microsomal preparations from developing castor bean (Ricinus communis L.).

The major b-type cytochrome in microsomal membrane preparations from developing endosperm of castor bean (Ricinus communis) was cytochrome b5. Cytochrome P-450 was also present. The microsomal membranes had delta 12-hydroxylase activity and catalysed the NAD(P)H-dependent hydroxylation of oleate to yield ricinoleic acid. CO had no effect on the hydroxylase activity. Rabbit polyclonal antibodies were raised against the hydrophilic cytochrome b5 fragment purified from cauliflower (Brassica oleracea) floret microsomes. The anti-(cytochrome b5) IgG inhibited delta 12-hydroxylase, delta 12-desaturase and cytochrome c reductase activity in the microsomes. The results indicate that electrons from NAD(P)H were transferred to the site of hydroxylation via cytochrome b5 and that cytochrome P-450 was not involved.

Ricinoleic acid biosynthesis and triacylglycerol assembly in microsomal preparations from developing castor-bean (Ricinus communis) endosperm.

Microsomal membrane preparations from the developing endosperm of castor bean (Ricinus communis) catalysed the transfer of oleate from [14C]oleoyl-CoA to phosphatidylcholine (PtdCho). In the presence of NADH, radioactive ricinoleate (12-hydroxyoctadec-9-enoate) was synthesized from [14C]oleate, and this was largely recovered in PtdCho and as free fatty acid. The addition of unlabelled ricinoleoyl-CoA to these incubation mixtures did not increase the low [14C]ricinoleate concentration found in the acyl-CoA fraction nor decrease the [14C]ricinoleate concentration in PtdCho and free fatty acid, and thus no evidence was obtained for a hydroxylation with oleoyl-CoA as a substrate. The addition of NADH, necessary for the formation of ricinoleate, caused a decrease of the total radioactivity in PtdCho with a corresponding increase in the amount of label in free ricinoleic acid. This increase was due to the action of a phospholipase A, which released ricinoleic acid but not oleic acid from PtdCho. Such a phospholipase activity, attacking ricinoleoyl-PtdCho but not oleoyl-PtdCho, was also demonstrated in microsomal preparations from developing cotyledons of safflower and oil-seed rape. An analysis of the acyl groups at different positions in microsomal PtdCho of castor bean showed that ricinoleate was almost entirely associated with position sn-2. Likewise the [14C]ricinoleate in [14C]PtdCho formed after incubations with microsomal preparations with NADH and [14C]oleoyl-CoA resided in position sn-2 with none in position sn-1. In contrast, the [14C]linoleate formed by desaturation of [14C]oleoyl-PtdCho was present at both positions. In the presence of ATP, CoA and Mg2+, the ricinoleate acid released from PtdCho was activated to ricinoleoyl-CoA. The ricinoleoyl-CoA was an efficient acyl donor in the acylation of glycerol 3-phosphate (Gro3P) to yield phosphatidic acid and triacylglycerols. In microsomal preparations incubated with an equimolar mixture of [14C]oleoyl-CoA and [14C]ricinoleoyl-CoA in the presence of Gro3P, only a minor amount of [14C]ricinoleate entered PtdCho, and this was believed to be via the exchange of phosphocholine groups between a diacylglycerol pool and the PtdCho. On the basis of our results, a scheme of ricinoleate formation and its incorporation into triacylglycerols in castor-bean endosperm is proposed.

Electron-transport components of the 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase (delta 12-desaturase) in microsomal preparations from developing safflower (Carthamus tinctorius L.) cotyledons.

The major cytochrome in microsomal membrane preparations from developing seeds of safflower (Carthamus tinctorius, var High Linoleate), has a reduced-minus-oxidized difference spectrum characteristic of a b-type cytochrome, and was identified from its midpoint-potential (E'7.2) value as cytochrome b5. Cytochromes P-450 and P-420 were also present. The cytochrome b5 content of microsomal preparations from a number of oilseed species was found to be in the order of 200-300 pmol/mg of protein. The cytochrome b5 was reduced in the membrane preparations by NADH, demonstrating the presence of an NADH: cytochrome b5 reductase; NADPH was a less effective donor. Microsomal membranes catalysed the NAD(P)H-dependent conversion of radioactive oleate into linoleate, indicating acyl-CoA: lysophosphatidylcholine acyltransferase and 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase (delta 12-desaturase) activity. Desaturation of oleate to linoleate was unaffected by CO, but inhibited by CN-. The addition of oleoyl-CoA to the NADH-reduced membranes resulted in the CN(-)-sensitive partial re-oxidation of cytochrome b5, indicating that electrons from NADH were transferred to the site of desaturation via this cytochrome. The delta 12-desaturase in safflower, therefore, is CN(-)-sensitive and appears to require cytochrome b5 and NADH: cytochrome b5 reductase for activity.