Biochemical, immunological, and in vivo functional characterization of B-domain-deleted factor VIII

Coagulation factor VIII (FVIII) is a cofactor in the intrinsic pathway of blood coagulation for which deficiency results in the bleeding disorder hemophilia A. FVIII contains a domain structure of A1-A2-B-A3- C1-C2 of which the B domain is dispensable for procoagulant activity in vitro. In this report, we compare the properties of B-domain-deleted FVIII (residues 760 through 1639, designated LA-VIII) to wildtype recombinant FVIII. In transfected Chinese hamster ovary (CHO) cells, LA- VIII was expressed at a 10- to 20-fold greater level compared with wildtype FVIII. The specific activity of purified LA-VIII was indistinguishable from wild-type recombinant FVIII and both exhibited similar thrombin activation coefficients. Wildtype recombinant-derived FVIII and LA-VIII also displayed similar timecourses of thrombin activation and heavy chain cleavage. However, compared with wildtype recombinant-derived FVIII, the light chain of LA-VIII was cleaved fivefold more rapidly by thrombin. Addition of purified von Willebrand factor (vWF) did not alter the kinetics of thrombin cleavage or activation of either wildtype recombinant-derived FVIII or LA-VIII. The immunogenicity of LA-VIII was compared with wildtype FVIII in a novel model of neonatal tolerance induction in mice. The results did not detect any immunologic differences between wildtype FVIII and LA-VIII, suggesting that LA-VIII does not contain significant new epitopes that are absent in wildtype FVIII. LA-VIII was tolerated well on infusion into FVIII-deficient dogs and was able to correct the cuticle bleeding time similar to wildtype recombinant factor VIII. In vivo, LA-VIII was bound to canine vWF and exhibited a half-life similar to wildtype recombinant FVIII. These studies support that B-domain-deleted FVIII may be efficacious in treatment of hemophilia A in humans.     

Anti-My-28, an antigranulocyte mouse monoclonal antibody, binds to a sugar sequence in lacto-N-neotetraose

Anti-My-28 is an IgM kappa monoclonal antibody produced by a hybridoma prepared from spleen cells of a mouse immunized with normal human granulocytes. By immunofluorescence it binds to human granulocytes but not to monocytes and lymphocytes. However, after treating cells with neuraminidase, the antibody also binds to lymphocytes and monocytes and to many leukemic cell lines and patient leukemic blast cells. Anti-My- 28 binds to several neutral glycolipids and desialylated gangliosides of leukocytes and erythrocytes as detected by radioimmunoassay and immunostaining of thin-layer chromatograms. It recognizes a sugar sequence in lacto-N-neotetraose, Gal beta 1-4GlcNAc beta 1-3Gal beta 1- 4Glc. This tetrasaccharide occurs in the glycolipids paragloboside and sialosylparagloboside, and its distal trisaccharide sequence is found in higher glycolipids and in glycoproteins.     

Dysglycemias in pregnancy: from diagnosis to treatment. Brazilian consensus statement

There is an urgent need to find consensus on screening, diagnosing and treating all degrees of DYSGLYCEMIA that may occur during pregnancies in Brazil, considering that many cases of DYSGLYCEMIA in pregnant women are currently not diagnosed, leading to maternal and fetal complications. For this reason the Brazilian Diabetes Society (SBD) and the Brazilian Federation of Gynecology and Obstetrics Societies (FEBRASGO), got together to introduce this proposal. We present here a joint consensus regarding the standardization of clinical management for pregnant women with any degree of Dysglycemia, on the basis of current information, to improve medical assistance and to avoid related complications of Dysglycemia in pregnancy to the mother and the fetus. This consensus aims to standardize the diagnosis among general practitioners, endocrinologists and obstetricians allowing the dissemination of information in basic health units, public and private services, that are responsible for screening, diagnosing and treating disglycemic pregnant patients.     

Safety and efficacy of liraglutide in patients with type 2 diabetes with elevated liver enzymes: individual patient data meta-analysis of the LEAD programme

BackgroundFatty liver disease has reached epidemic proportions in type 2 diabetes. Glucagon-like peptide-1 (GLP-1) analogues are licensed for treatment of type 2 diabetes, yet little data exist on efficacy and safety in liver injury. We aimed to assess the safety and efficacy of 26 weeks' liraglutide on liver function compared with an active placebo.MethodsIndividual patient data meta-analysis was done with patient level data combined from six 26-week, phase 3, double-blind randomised controlled trials on type 2 diabetes, which comprise the Liraglutide Effect and Action in Diabetes (LEAD) programme. In addition, the LEAD-2 sub-study was analysed to assess the effect on CT-measured hepatic steatosis.FindingsOf 4442 patients analysed, 2241 (50·8%) had an abnormal alanine aminotransferase (ALT) at baseline (mean 33·8 IU/L [SD 14·9] in female participants; 47·3 [18·3] in male participants). Liraglutide 1·8 mg reduced ALT in these patients compared with placebo (?8·20 vs ?5·01 IU/L, p=0·003), and was dose dependent (no significant differences vs placebo with liraglutide 0·6 or 1·2 mg). This effect was lost after adjustment for liraglutide's effect on reduction of weight (corrected mean ALT difference vs placebo ?1·41 IU/L, p=0·21) and HbA1c (corrected mean ALT difference vs placebo 0·57 IU/L, p=0·63). Adverse effects with 1·8 mg liraglutide were similar between patients with and without baseline abnormal ALT. In the LEAD-2 sub-study, liraglutide 1·8 mg (26 weeks) improved hepatic steatosis (CT-measured liver:spleen attenuation ratio) from baseline (0·10, p=0·001) and showed a trend towards improvement compared with placebo (0.10 vs 0·00, p=0·07).Interpretation26 weeks of liraglutide (1·8 mg) is safe, well tolerated, and improves liver enzymes compared with placebo in patients with type 2 diabetes.FundingWellcome Trust.     

Ricinus communis Agglutinin I Leads to Rapid Down-Regulation of VEGFR-2 and Endothelial Cell Apoptosis in Tumor Blood Vessels

Ricinus communis agglutinin I (RCA I), a galactose-binding lectin from castor beans, binds to endothelial cells at sites of plasma leakage, but little is known about the amount and functional consequences of binding to tumor endothelial cells. We addressed this issue by examining the effects of RCA I on blood vessels of spontaneous pancreatic islet-cell tumors in RIP-Tag2 transgenic mice. After intravenous injection, RCA I bound strongly to tumor vessels but not to normal blood vessels. At 6 minutes, RCA I fluorescence of tumor vessels was largely diffuse, but over the next hour, brightly fluorescent dots appeared as the lectin was internalized by endothelial cells. RCA I injection led to a dose- and time-dependent decrease in vascular endothelial growth factor receptor-2 (VEGFR-2) immunoreactivity in tumor endothelial cells, with 95% loss over 6 hours. By comparison, VEGFR-3, CD31, and CD105 had decreases in the range of 21% to 33%. Loss of VEGFR-2 was followed by increased activated caspase-3 in tumor vessels. Prior inhibition of VEGF signaling by AG-028262 decreased RCA I binding and internalization into tumor vessels. These findings indicate RCA I preferentially binds to and is internalized by tumor endothelial cells, which leads to VEGFR-2 down-regulation, endothelial cell apoptosis, and tumor vessel regression. Together, the results illustrate the selective impact of RCA I on VEGF signaling in tumor blood vessels.Tumor vessels are irregularly shaped and tortuous, and have multiple functional abnormalities.^1,2,3 Endothelial cells comprising tumor vessels have abnormalities in gene expression, require growth factors for survival, and have defective barrier function to plasma proteins.^1,3 Molecules that are preferentially expressed on tumor vessels can serve as therapeutic targets.²Plant lectins have been used to characterize the surface properties of cells and to isolate membrane proteins of endothelial cells.^4,5,6,7,8 Ricinus communis agglutinin (RCA I, RCA 120) and Ricinus communis toxin (ricin, RCA II, RCA 60) are galactose-binding lectins from seeds of the castor bean plant R. communis.^{9,10,11,12,13} Both lectins bind to erythrocytes and endothelial cells in vitro,^14,15,16 but the effects are strikingly different. RCA I is a potent galactoside-binding lectin and hemagglutinin, and ricin is a potent enzymatically active toxin.¹¹RCA I and ricin are heterodimeric proteins with two components. RCA I consists of two A chains and two B chains, and ricin has one A and one B chain joined by a single disulfide bridge.^{9,10,11,12,13} The sequence of the A and B chains of RCA I and ricin have homologies but are not identical.¹² The A chain of RCA I and ricin is cytotoxic because its glycosidase activity inactivates ribosomal protein synthesis; the B chain is a lectin that mediates endocytosis by binding galactosyl and N-acetylgalactosaminyl residues of cell surface glycoconjugates.^12,13 However, ricin (LD₅₀ 5 to 36 μg/kg in mice) is much more toxic than RCA I (LD₅₀ ∼1400 μg/kg).^17,18 A single molecule of enzymatically active ricin A chain reportedly can inactivate 1500 ribosomes per minute, inhibit protein synthesis, and cause cell death.¹³ Because of this activity, ricin has been used as a biological weapon and as an immunotoxin for cancer therapeutics.^{13,19,20,21,22,23} Effects of ricin by endothelial cells may result in the vascular leak syndrome found with immunotoxins or other therapeutics containing ricin A chain.^24,25,26,27Studies of lectins injected into the vasculature have shown that RCA I, unlike Lycopersicon esculentum (LEA, tomato lectin), does not bind uniformly to the luminal surface of the endothelium, but instead binds preferentially to leaky sites in the endothelium of inflamed venules.⁷ RCA I also binds to the luminal surface of endothelial cells of murine squamous carcinomas, indicative of affinity for tumor blood vessels,²⁸ and to sinusoidal endothelial cells of liver and bone marrow²⁹ and certain other vessels.³⁰To obtain a better understanding of the selectivity and functional consequences of RCA I binding to endothelial cells of tumor blood vessels in vivo, we examined the distribution of rhodamine-labeled RCA I in tumors after i.v. injection into RIP-Tag2 transgenic mice that have spontaneous pancreatic islet cell adenomas and carcinomas. Using immunohistochemistry and fluorescence and confocal microscopy, we found that rhodamine-RCA I bound much more strongly to tumor vessels than to normal blood vessels of the surrounding acinar pancreas. The lectin initially coated much of the luminal surface of tumor vessels but after an hour had a conspicuous dot-like pattern, indicative of internalization into endosomes and lysosomes of endothelial cells. Vascular endothelial growth factor (VEGF) receptor (R)-2 immunoreactivity of endothelial cells subsequently decreased, endothelial cells underwent apoptosis, and tumor vessels regressed. These findings demonstrate that RCA I can selectively reduce VEGFR-2 in endothelial cells of tumor blood vessels in vivo.     

SU14813: a novel multiple receptor tyrosine kinase inhibitor with potent antiangiogenic and antitumor activity

Receptor tyrosine kinases (RTK), such as vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), stem cell factor receptor (KIT), and fms-like tyrosine kinase 3 (FLT3), are expressed in malignant tissues and act in concert, playing diverse and major roles in angiogenesis, tumor growth, and metastasis. With the exception of a few malignancies, seemingly driven by a single genetic mutation in a signaling protein, most tumors are the product of multiple mutations in multiple aberrant signaling pathways. Consequently, simultaneous targeted inhibition of multiple signaling pathways could be more effective than inhibiting a single pathway in cancer therapies. Such a multitargeted strategy has recently been validated in a number of preclinical and clinical studies using RTK inhibitors with broad target selectivity. SU14813, a small molecule identified from the same chemical library used to isolate sunitinib, has broad-spectrum RTK inhibitory activity through binding to and inhibition of VEGFR, PDGFR, KIT, and FLT3. In cellular assays, SU14813 inhibited ligand-dependent and ligand-independent proliferation, migration, and survival of endothelial cells and/or tumor cells expressing these targets. SU14813 inhibited VEGFR-2, PDGFR-beta, and FLT3 phosphorylation in xenograft tumors in a dose- and time-dependent fashion. The plasma concentration required for in vivo target inhibition was estimated to be 100 to 200 ng/mL. Used as monotherapy, SU14813 exhibited broad and potent antitumor activity resulting in regression, growth arrest, or substantially reduced growth of various established xenografts derived from human or rat tumor cell lines. Treatment in combination with docetaxel significantly enhanced both the inhibition of primary tumor growth and the survival of the tumor-bearing mice compared with administration of either agent alone. In summary, SU14813 inhibited target RTK activity in vivo in association with reduction in angiogenesis, target RTK-mediated proliferation, and survival of tumor cells, leading to broad and potent antitumor efficacy. These data support the ongoing phase I clinical evaluation of SU14813 in advanced malignancies.     

Specificity of mutagenesis by 4-aminobiphenyl: mutations at G residues in bacteriophage M13 DNA and G-->C transversions at a unique dG(8-ABP) lesion in single-stranded DNA

Mutagenesis by the human bladder carcinogen 4-aminobiphenyl (ABP) was studied in single-stranded DNA from a bacteriophage M13 cloning vector. In comparison to ABP lesions in double-stranded DNA, lesions in single- stranded DNA were approximately 70-fold more mutagenic and 50-fold more genotoxic. Sequencing analysis of ABP-induced mutations in the lacZ gene revealed exclusively base-pair substitutions, with over 80% of the mutations occurring at G sites; the G at position 6310 accounted for 25% of the observed mutations. Among the sequence changes at G sites, G- ->T transversions predominated, followed by G-->C transversions and G-- >A transitions. In order to further elucidate the mutagenic mechanism of ABP, an oligonucleotide containing the major DNA adduct, N- (deoxyguanosin-8-yl)-4-aminobiphenyl (dG(8-ABP)), was situated within the PstI site of a single-stranded M13 genome. After in vivo replication of the adduct containing ABP-modified and control (unadducted) genomes, the mutational frequency and mutational specificity of the dG(8-ABP) lesion were determined. The targeted mutational efficiency was approximately 0.01%, and the primary mutation observed was the G-->C transversion. Thus dG(8-ABP), albeit weakly mutagenic at the PstI site, can contribute to the mutational spectrum of ABP lesions.      

The survival motor neuron protein in spinal muscular atrophy

The 38 kDa survival motor neuron (SMN) protein is encoded by two ubiquitously expressed genes: telomeric SMN (SMN(T)) and centromeric SMN (SMN(C)). Mutations in SMN(T), but not SMN(C), cause proximal spinal muscular atrophy (SMA), an autosomal recessive disorder that results in loss of motor neurons. SMN is found in the cytoplasm and nucleus. The nuclear form is located in structures termed gems. Using a panel of anti-SMN antibodies, we demonstrate that the SMN protein is expressed from both the SMN(T) and SMN(C) genes. Western blot analysis of fibroblasts from SMA patients with various clinical severities of SMA showed a moderate reduction in the amount of SMN protein, particularly in type I (most severe) patients. Immunocytochemical analysis of SMA patient fibroblasts indicates a significant reduction in the number of gems in type I SMA patients and a correlation of the number of gems with clinical severity. This correlation to phenotype using primary fibroblasts may serve as a useful diagnostic tool in an easily accessible tissue. SMN is expressed at high levels in brain, kidney and liver, moderate levels in skeletal and cardiac muscle, and low levels in fibroblasts and lymphocytes. In SMA patients, the SMN level was moderately reduced in muscle and lymphoblasts. In contrast, SMN was expressed at high levels in spinal cord from normals and non- SMA disease controls, but was reduced 100-fold in spinal cord from type I patients. The marked reduction of SMN in type I SMA spinal cords is consistent with the features of this motor neuron disease. We suggest that disruption of SMN(T) in type I patients results in loss of SMN from motor neurons, resulting in the degeneration of these neurons.      

Preimplantation hormonal differences between the conception and non- conception menstrual cycles of 32 normal women

We compared daily urinary concentrations of oestrogen and progesterone metabolites in paired menstrual cycles (conception and non-conception) from 32 women. Volunteers with no known fertility problems were enrolled in the study at the time they began trying to become pregnant. They collected first-morning urine specimens and kept daily records of menstrual bleeding and sexual intercourse for 6 months or until they became clinically pregnant. Intercourse in non-conception cycles was close to the time of ovulation so that failure to conceive was caused by factors other than poorly timed intercourse. Compared with non- conception cycles, conception cycles had a steeper early luteal rise in progesterone and higher mid-luteal oestrogen and progesterone concentrations. These hormonal characteristics may be markers of better quality cycles, but because all these differences were in the luteal phase, we cannot rule out the possibility that the preimplantation embryo had stimulated early increases in steroid production. We propose an analysis strategy that could help support or refute the importance of preimplantation embryonic signalling, but our small sample size limits our own conclusions about this mechanism.