A prospective surveillance study of inhibitor development in haemophilia A patients following a population switch to a third-generation B-domain-deleted recombinant factor VIII.

INTRODUCTION: Following a provincial tender, most subjects with haemophilia A in Quebec switched their treatment to a third-generation recombinant B-domain-deleted factor VIII (FVIII). AIM: Our objective was to evaluate the incidence of inhibitor development and FVIII recovery in patients following the switch of factor replacement therapy. METHODS: One hundred and thirty-five subjects were enrolled and tested for FVIII activity and inhibitors every 6 months during 1 year. Subjects with mild haemophilia A or current inhibitors were excluded. Data on demographics, bleeds and FVIII usage were collected. RESULTS: A total of 125 switchers and 10 non-switchers were enrolled. Most subjects had severe haemophilia A (95.6%) and were on prophylaxis (89.6%). Mean FVIII recovery was similar at 0, 6 and 12 months postswitch. Two switchers developed de novo inhibitors in the 6 months postswitch, one of which was transient. No recurrent inhibitor was observed. A small but significant increase in FVIII usage was observed for adult switchers and the whole cohort of switchers and non-switchers. There was an increase in the annualized bleeding rate (ABR) for non-joint bleeds for the whole cohort of switchers. However, no significant differences were observed in ABR for joint bleeds. CONCLUSION: Our surveillance study shows comparable inhibitor development to similar published studies. A significant increase in FVIII utilization was noted for the whole cohort, switchers and non-switchers. Lastly, no clinically significant changes were observed in ABR for joint bleeds, but a difference for non-joint bleed ABRs was observed in switchers.

Alloantibody developed in a factor XIII A subunit deficient patient during substitution therapy; characterization of the antibody.

INTRODUCTION: In factor XIII A subunit (FXIIIA) deficiency, the development of alloantibodies is extremely rare. Only four reports have been published and the antibodies were not characterized. AIM: The aim of this study was to describe the clinical course and the laboratory diagnosis of a FXIII-A deficient patient who developed alloantibodies. METHODS: FXIII activity was assessed with an ammonia release assay. FXIII-A, FXIII B subunit (FXIII-B) and the complex plasma FXIII (FXIII-A2 B2 ) antigens were determined by ELISA. The causative mutation was detected by fluorescent DNA sequencing. The binding of alloantibody to FXIII-A2 and FXIII-A2 B2 was studied by surface plasmon resonance. The cleavage of FXIII-A by thrombin and Ca2+ -induced activation of thrombin-cleaved FXIII were followed by western blotting and activity measurement, respectively. RESULTS: FXIII activity, FXIII-A2 B2 and FXIII-A antigens were below the limit of detection in the patient's plasma. The severe FXIII-A deficiency was due to a novel homozygous mutation resulting in early stop codon (c.127C>T, p.Gln42STOP). The alloantibody bound to FXIII-A2 and FXIII-A2 B2 with equally high affinity (Kd ~10-8 ). It accelerated the elimination of administered FXIII concentrate from the circulation, interfered with thrombin and Ca2+ -induced activation and inhibited FXIII activity. Attempts to eliminate the alloantibody resulted only in transient improvement. Patient developed intracerebral haemorrhage after a minor trauma and died in spite of aggressive replacement therapy with FXIII concentrate. CONCLUSION: The anti-FXIII-A alloantibody caused an unmanageable bleeding complication. The antibody was of combined subtype which accelerated the elimination of FXIII and exerted a multiple inhibitory effect on FXIII activation/activity.

Incidence and risk factors for inhibitor development in previously untreated severe haemophilia A patients born between 2005 and 2010.

The objective of this study was to evaluate the inhibitor development (ID) in previously untreated patients (PUPs) with severe haemophilia A (FVIII ≤ 0.01 IU mL(-1) ). All Canadian Haemophilia Treatment Centres completed a questionnaire on patients born between September 2005 and August 2010 and followed for up to 7 years. Eligible patients had at least 20 exposure days (ED) or had developed an inhibitor. The odds ratio (OR) and 95% confidence intervals (95% CI) for risk factors to develop an inhibitor were estimated using unconditional logistic regression. A total of 99 haemophilia A PUPs were studied. Thirty-four (34%) developed an inhibitor (24/34 of high titre). Inhibitors developed in 25/63 (40%) patients with a high-risk mutation. ID was most frequent in Aboriginals (86%). Dose intensity (IU kg(-1)  day(-1) X number of ED) at first exposure to factor VIII (FVIII) was associated with a crude OR increase of 1.10 (95% CI: 0.99-1.23) with each increase of 100 dose-intensity units. Haemarthrosis and intracranial bleeding as the indication for first exposure to FVIII concentrate were associated with a crude OR for ID of 7.63 (95% CI: 2.14-27.17) and 5.08 (95% CI: 1.11-23.31) respectively. ID according to FVIII concentrate used was: Advate (®) 18/50 (36%), Kogenate FS(®) or Helixate FS(®) 15/36 (42%), Wilate(®) 0/11 and Xyntha(®) 1/2. In multivariate analysis, Aboriginal ethnicity (OR = 11.69; 95% CI: 1.11-122.86) and haemarthrosis (OR = 4.49; 95% CI: 1.08-18.61) were statistically significant. The cumulative incidence of ID in severe haemophilia A PUPs was 34% and varied according to ethnicity, type of bleeding at first ED, type of FVIII product and dose intensity at first exposure.

Apolipoprotein A-I Q[-2]X causing isolated apolipoprotein A-I deficiency in a family with analphalipoproteinemia.

We report a Canadian kindred with a novel mutation in the apolipoprotein (apo) A-I gene causing analphalipoproteinemia. The 34-yr-old proband, product of a consanguineous marriage, had bilateral retinopathy, bilateral cataracts, spinocerebellar ataxia, and tendon xanthomata. High density lipoprotein cholesterol (HDL-C) was < 0.1 mM and apoA-I was undetectable. Genomic DNA sequencing of the proband's apoA-I gene identified a nonsense mutation at codon [-2], which we designate as Q[-2]X. This mutation causes a loss of endonuclease digestion sites for both BbvI and Fnu4HI. Genotyping identified four additional homozygotes, four heterozygotes, and two unaffected subjects among the first-degree relatives. Q[-2]X homozygosity causes a selective failure to produce any portion of mature apoA-I, resulting in very low plasma level of HDL. Heterozygosity results in approximately half-normal apoA-I and HDL. Gradient gel electrophoresis and differential electroimmunodiffusion assay revealed that the HDL particles of the homozygotes had peak Stokes diameter of 7.9 nm and contained apoA-II without apoA-I (Lp-AII). Heterozygotes had an additional fraction of HDL3-like particles. Two of the proband's affected sisters had documented premature coronary heart disease. This kindred, the third reported apoA-I gene mutation causing isolated complete apoA-I deficiency, appears to be at significantly increased risk for atherosclerosis.

Externalization of host cell protein kinase C during enteropathogenic Escherichia coli infection.

Enteropathogenic Escherichia coli (EPEC) is a common cause of diarrhea in children in developing countries. Protein kinase C (PKC), a serine- and threonine-directed protein kinase, is rapidly activated following EPEC infection and this is accompanied by its translocation to a membrane-bound location where it is tightly bound to phosphatidylserine (PS). EPEC infection causes host cell death, one of whose features is externalization of PS. We hypothesized that externalization of PS would be accompanied by externalization of PKC as well. We report that EPEC infection triggers the externalization of PKC to the outer surface of the host cell. Ecto-PKC remains firmly tethered to the cell but can be released by incubation with peptide or protein substrates for the enzyme. Ecto-PKC is intact and biologically active and able to phosphorylate protein substrates on the surface of the host cell. Phosphorylation of whole EPEC bacteria or EPEC-secreted proteins could not be detected. Externalization of PKC could be reproduced by the combination of an apoptotic stimulus (ultraviolet (UV) irradiation) and phorbol myristate acetate (PMA), a procedure which resulted in externalization of >25% of the total cellular content of PKC-alpha. In the presence of ATP, ecto-PKC inhibited UV-induced cell shrinkage, membrane blebbing, and propidium iodide uptake but not the activation of caspases 3 and 7. This is the first report that expression of an ecto-protein kinase is altered by a microbial pathogen and the first to note that externalization of PKC can accompany apoptosis.

Cholesteryl ester and apolipoprotein E transfer between human high density lipoproteins and chylomicrons.

The transfer of cholesteryl esters and apolipoprotein E has been studied between plasma HDL and chylomicrons isolated either from ascitic fluid or from the plasma of a patient with type V hyperlipoproteinemia. Whereas apolipoprotein E transfer was rapid and occurred at low temperature, cholesteryl ester transfer was suppressed at 4 degrees C. Apolipoprotein E transfer did not depend upon the presence of cholesteryl ester transfer protein and was in fact inhibited by the partially purified preparation of this protein. Apolipoprotein E transfer was not increased by reduction with dithiothreitol. The transfer of cholesteryl esters increased sharply at a chylomicron to HDL ratio of cholesteryl ester above 1/10, a value which may be of physiological significance at the peak of postprandial lipemia. At this ratio, the transfer of apolipoprotein E was minimal and increased only at ratios above 2/1. From these results, it is concluded that there is no connection between apolipoprotein E and cholesteryl ester transfer from HDL to chylomicrons. It is, therefore, proposed that whereas chylomicron apolipoprotein E is acquired rapidly and mostly in the lymphatic system, the concentration of chylomicron cholesteryl esters increases significantly and independently in the circulation.

Comparison of very-low-density lipoproteins isolated from rat liver perfusate, rat serum and human plasma as acceptors for cholesteryl ester transfer.

Using two methods, we have compared the ability of three types of very-low-density lipoprotein (VLDL), namely, rat hepatic perfusate VLDL, rat serum VLDL and human plasma VLDL, to accept cholesteryl esters from human plasma high-density lipoprotein (HDL). Both net mass and isotopic transfers of cholesteryl ester were evaluated. The VLDL concentration in the incubation media was adjusted on the basis of equivalent amounts of either cholesteryl ester, apolipoprotein B or triacylglycerol. In each case, the net mass transfer of cholesteryl ester was found to be greatest to rat hepatic VLDL, lower to rat serum VLDL and lowest to human plasma VLDL. Similar results were obtained with the isotopic transfer method when the VLDL concentration was adjusted on the basis of cholesteryl ester or apolipoprotein B. However, when VLDL were matched on the basis of triacylglycerol concentration, the isotopic transfer rate into human plasma VLDL was twice as high as into rat hepatic VLDL which presumably reflects the decreased number of hepatic VLDL particles by this criterion. In conclusion under fasted conditions (chylomicron excluded), nascent VLDL have been shown to be the best cholesteryl ester acceptor compared to their plasma counterpart.

A hepatic lipase gene mutation associated with heritable lipolytic deficiency.

Absent hepatic lipase (HL) activity results in dyslipidemia and premature atherosclerosis. DNA sequencing of the HL gene from subjects with heritable HL deficiency identified a new C to T substitution within exon 8 that in the mature enzyme caused a threonine to methionine change at position 383 (T383M). With a rapid DNA detection method we observed that all 6 individuals with complete HL deficiency from 2 families had the T383M mutation. None of 50 random unrelated unaffected subjects had this mutation. We propose that T383M is specific to families with heritable HL deficiency. Furthermore, structural variation at the HL gene, possibly in combination with other factors, appears to be etiologic in HL deficiency.

Specific types of colonic fermentation may raise low-density-lipoprotein-cholesterol concentrations.

To assess the effects of increased colonic fermentation on serum lipids, eight healthy volunteers were placed on two identical 2-wk metabolic diets, one of which was supplemented with lactulose (18-25 g/d). Lactulose raised day-long concentrations of breath hydrogen and serum glutamine as indicators of increased colonic fermentation by 78 +/- 13% (P less than 0.001) and 24.7 +/- 9.5% (P less than 0.05), respectively). Unexpectedly, however, fasting serum total and low-density-lipoprotein cholesterol and apolipoprotein B concentrations were higher at 2 wk by 8.9 +/- 1.5% (P less than 0.001), 10.9 +/- 2.2% (P less than 0.005), and 18.9 +/- 5.9% (P less than 0.02), respectively, compared with the control diet. With lactulose, mean free fatty acid concentrations were reduced over the day by 19.5 +/- 5.9% (P less than 0.02), with no change in mean day-long blood glucose, serum insulin, or C-peptide concentrations. We conclude that certain rapidly fermented substrates may raise rather than lower serum lipids, possibly through increasing the amount of acetate absorbed from the colon.

Transfer of cholesterol esters between human high density lipoproteins and triglyceride-rich lipoproteins controlled by a plasma protein factor.

A protein factor from the d greater than 1.25 g/ml plasma fraction controls the transfer of cholesterol esters between high density lipoproteins and very low density lipoproteins. This transfer is time-dependent, and follows saturation kinetics relative to the concentration ratio of acceptor to donor lipoproteins. Although the process is reversible, the transfer rates are faster from high density to very low density lipoproteins and result in a net increase of cholesterol esters in the very low density lipoproteins. Under the same conditions, there is also a net mass transfer of cholesterol esters from high density lipoproteins to chylomicrons. This constitutes the first demonstration of cholesterol ester mass transfer between isolated lipoproteins and contrasts with the equilibrium of cholesterol esters between HDL and LDL which we previously demonstrated [4]. The apparent maximum transfer rate of cholesterol esters from high density to very low density lipoproteins was calculated to be about 80 nmoles cholesterol esters/h/ml plasma, which is very similar to the initial rate of reaction of lecithin:cholesterol acyltransferase in plasma. It is concluded that cholesterol ester formation in high density lipoproteins and their transfer to triglyceride-rich lipoproteins may be closely coupled.

Cholesterol ester exchange between human plasma high and low density lipoproteins mediated by a plasma protein factor.

Although unesterified cholesterol and phospholipids exchange freely, a protein factor from the d greater than 1.25 g/ml plasma fraction was found to be necessary for cholesterol esters to transfer from HDL to LDL. This transfer was reversible, time-dependent and a function of the concentration of the d greater than 1.25 fraction, but independent of lecithin : cholesterol acyltransferase reaction. The transfer represented an equilibration of molecules, but no net mass transfer of cholesterol esters could be demonstrated from HDL to LDL.

Lecithin: cholesterol acyltransferase activity and fatty acid composition of erythrocyte phospholipids in Friedreich's ataxia.

In a study of the fatty acid composition of erythrocyte membrane phospholipids in Friedreich's ataxia, a lower percentage of linoleic acid in phosphatidylcholine was demonstrated. An enzyme involving the exchange of lipids between plasma and erythrocyte membrane, lecithin: cholesteryl acyltransferase (LCAT) was also studied. It was found that the LCAT activity had a trend towards low values. However, crossing-over studies indicated that when the LCAT enzyme of patients was exposed to its own substrate it gave low activity values but that the result reverted to normal when control substrate was used.

Rapamycin (AY-22,989), a new antifungal antibiotic. IV. Mechanism of action.

Rapamycin, an antifungal antibiotic produced by Streptomyces hygroscopicus showed a strong candicidal activity, which could not be reversed by sterols. It has no effect on efflux of K+, Pi ir U.V. absorbing materials and cell permeability of Candida albicans. Thus, in its action it differs from the polyenes. Mechanism of action of rapamycin appears to be different from many known antifungal agents. In C. albicans, rapamycin at the minimum growth inhibitory concentration inhibited: 1) phosphate incorporation into nucleic acids, 2) acetate incorporation into lipids and 3) substrate respiration of amino acids. The effect on amino acid metabolism was expressed as inhibition of oxidative deamination. At low concentrations rapamycin caused degradation of P32-labeled intracellular macromolecules. Inhibition of threonine incorporation into cell wall and leucine incorporation into cellular protein was observed at relatively higher concentrations of rapamycin. The antibiotic showed no effect on cell-free protein synthesizing systems of Escherichia coli, rat liver and C. albicans and in the mitochondrial enzyme systems. Whether the lethal action of rapamycin on C. albicans is primarily due to one of the above effects or is the result of combined effect on some of these biosynthetic parameters remains to be established.

Activity of rapamycin (AY-22,989) against transplanted tumors.

Rapamycin exhibits activity against several ascites and solid transplantable tumors; it is slightly active to inactive against leukemias. On a weight basis, rapamycin was less active than 5-fluorouracil, cyclophosphamide and adriamycin, but rapamycin's maximal activity against Colon 38 tumor was similar to that of 5-fluorouracil and cyclophosphamide. Its activity was such that it significantly inhibited tumor growth at any stage of development. In the active dose range, rapamycin appeared less toxic than the other drugs. In the Colon 38 tumor model, rapamycin at a given dose exhibited the same activity when administered ip, iv, im and sc; upon oral administration, its activity was reduced but not abolished. Rapamycin was compatible with 5-fluorouracil and cyclophosphamide. The sequential treatment 5-fluorouracil-rapamycin-cyclophosphamide was superior to the sequence 5-fluorouracil-adriamycin-cyclophosphamide in protecting Colon 38 tumor-bearing mice. 29-Demethoxyrapamycin exerted only marginal activity against P388 lymphocytic leukemia; it was inactive against B16 melanocarcinoma and Colon 38 solid tumor.

Rapamycin (AY-22,989), a new antifungal antibiotic. I. Taxonomy of the producing streptomycete and isolation of the active principle.

A streptomycete was isolated from an Easter Island soil sample and found to inhibit Candida albicans, Microsporum gypseum and Trichophyton granulosum. The antibiotic-producing microorganism was characterized and identified as Streptomyces hygroscopicus. The antifungal principle was extracted with organic solvent from the mycelium, isolated in crystalline form and named rapamycin. Rapamycin is mainly active against Candida albicans; minimum inhibitory concentration against ten strains ranged from 0.02 to 0.2 mug/ml. Its apparent activity against Microsporum gypseum and Trichophyton granulosum is lower because of its instability in culture media on prolonged incubation required by these fungi. No activity was observed against gram-positive and gram-negative bacteria. Acute toxicity in mice is low.

Rapamycin (AY-22,989), a new antifungal antibiotic. II. Fermentation, isolation and characterization.

Rapamycin is a new antifungal antibiotic produced by Streptomyces hygroscopicus NRRL 5491. It was isolated from the mycelium by solvent extraction, purified by silica gel column chromatography and crystallized as a colorless solid which melts at 183 approximately to 185 degrees C and has the empirical formula C56H89NO14. From its characteristic ultraviolet absorption spectrum rapamycin can be classified as a triene. It is highly active against various Candida species, especially Candida albicans. Its activity is compared with that of amphotericin B, candicidin and nystatin.

Antimycin A fermentation. II. Fermentation in aerated-agitated fermenters.

Fermentation characteristics, previously studied in shake flasks, were reproduced in aerated-agitated fermenters, using three strains of Streptomyces sp. which had been selected for their high antimycin A productivity in shake flasks. Fermentation in fermenters was run in three stages. The medium consisted of soy flour, glucose, ammonium sulfate and calcium carbonate; initial pH was 7.2 approximately 7.5, and temperature 25 degrees C. The course of fermentation was then modified to encourage maximal growth and eliminate the intermediate lag period observed in shake flasks. Useful corrections included continuous addition of soybean oil at 1.25 %/day and maintenance of pH at 6 by addition of ammonium hydroxide on demand. The ammonium hydroxide added also served as a rapidly utilized nitrogen source and could not be replace by NaOH or KOH. Under optimal conditions antimycin A was produced at constant rate from the second to the sixth day, when maximum yields of more than 9 g/liter were attained. A procedure for antimycin A extraction is described.

Antimycin A fermentation. I. Production and selection of strains.

Increase in antimycin A production was achieved through a parallel strain and medium improvement program: a 125-fold augmentation (75 to 9,500 mug/ml) was obtained. The selective system included antimycin A productivity, conidiation, sensitivity to ultraviolet radiation, growth rate and yield, and absence of pigment and actinomycin D production. Among the original strains tested one natural isolate possessed high productivity and several of the above characteristics, and was selected for mutagenesis. Spontaneous and induced variability was then exploited in isolating high-producing strains. The first mutagen used was ultraviolet radiation; it was replaced by ethylenimine when it became no longer efficient in increasing variability. As new, high producers were isolated, the medium was modified to best suit their requirements for still higher productivity. The critical environmental factors were absence of phosphate and organic salts, concentration of the nitrogen source and ratio organic/inorganic nitrogen, ratio ammonium sulfate/calcium carbonate, and addition of slowly utilizable carbon sources, such as lactose and oil; optimum temperature and initial pH were 25 degrees C and 7.0. Aeration/agitation requirements of improved strains were high. Fermentation was characterized by abrupt pH changes which impaired rapid accumulation of the antibiotic. Antimycin A was produced during both the trophophase and idiophase.

Rapamycin (AY-22,989), a new antifungal antibiotic. III. In vitro and in vivo evaluation.

The activity of rapamycin, a new anti-Candida antibiotic, was not affected by pH values between 6 and 8; at pH 4, however, activity was abolished. The MIC of rapamycin did not vary drastically with the size of inoculum: a ten-fold dilution of the inoculum reduced the MIC only two-fold. Serum binding was extensive. Serum levels obtained in mice were higher on subcutaneous injection than with oral administration. Dogs absorbed rapamycin after oral administration. Rapamycin cured systemic candidosis in mice: PD50 s.c. was 9.5 mg/kg: PD50 p.o. was 11 mg/kg. In the same experimental infections amphotericin B and nystatin exhibited PD50 values of less than 0.25 mg and greater than 4,000 units/kg respectively. Rapamycin and amphotericin B, administered at 1, 4 and 24 hours after infection, gave approximately the same percent survival after 30 days of observation. When the above treatment was extended by an additional daily treatment for 6 days, rapamycin by the subcutaneous route yielded a higher percentage of survival than either rapamycin or amphotericin B, administered orally, after a 30-day observation period. Vaginal candidosis in female rats was treated efficiently (91% cure) by rapamycin administered orally. No increase of resistance of C. albicans was observed during treatment.

Naphthyridinomycin, a new broad-spectrum antibiotic.

A new antibiotic, naphthyridinomycin, was isolated in crystalline form from the culture filtrate of Streptomyces lusitanus AY B-1026. The antibiotic is active against a large number of both gram-positive and gram-negative bacteria, and inactive against Candida albicans, Trichophyton granulosum and Microsporum gypseum. The antibiotic is toxic in mice.