Studies on the mechanism of bacterial resistance to complement-mediated killing. II. C8 and C9 release C5b67 from the surface of salmonella minnesota S218 because the terminal complex does not insert into the bacterial outer membrane

The mechanism for consumption of terminal complement components and release of bound components from the surface of serum-resistant salmonella minnesota S218 was studied. Consumption of C8 and C9 by S218 occurred through interaction with C5b67 on the bacterial surface because C8 and C9 were consumed when added to S218 organisms previously incubated in C8-deficient serum and washed to remove all C5b67 on the bacterial surface because C8 and C9 were consumed when added to S218 organisms previously incubated in C8- deficient serum and washed to remove al but cell bound C5b67. Rapid release of (125)I C5 and (125)I C7 from the membrane of S218 was dependent on binding of C8 because (125)I C5 and (125)I C7 deposition in C8D serum was stable and was twofold higher in C8D than in PNHA, and addition of purified C8 or C8 and C9 to S218 previously incubated in C8D serum caused rapid release of (125)I C5 and (125)I C7 from the organism. Analysis by sucrose density gradient ultracentrifugation of the fluid phase from the reaction of S218 and 10 percent PNHS revealed a peak consistent with SC5b-9, in which the C9:C7 ratio was 3.3:1, but the NaDOC extracted bound C5b-9 complex sedimented as a broad peak with C9:C7 of less than 1.2:1. Progressive elution of C5b67 and C5b-9 from S218 but not serum-sensitive S. minnesota Re595 was observed with incubation in buffers of increasing ionic strength. Greater than 90 percent of the bound counts of (125)I C5 or (125)I C9 were released from S218 by incubation in 0.1 percent trypsin, but only 57 percent of (125)I C9 were released by treatment of Re595 with trypsin. These results are consistent with the concept that C5b-9 forms on the surface of the serum-sensitive S. minnesota S218 in normal human serum, but the formed complex is released and is not bactericidal for S218 because it fails to insert into hydrophobic outer membrane domains.     

Complement activation by artificial blood substitute Fluosol: in vitro and in vivo studies

A perfluorocarbon blood substitute, Fluosol, is undergoing clinical trials as an adjunct to chemotherapy. The adverse effects associated with its administration have been postulated to result from complement activation. When gel electrophoresis and Western blotting of Fluosol are used after its incubation with serum, activated C3 and factors Bb and H are bound to the Fluosol particles in a time-dependent fashion, which suggests that complement activation with Fluosol, as does that with zymosan, occurs on the surface of the particles. Paradoxically, it is found, both by the measurement of Fluosol-bound C3d and by fluid-phase C5a, that lower concentrations of Fluosol cause greater amounts of complement activation, which suggests a complex interaction of activators and inhibitors that changes as the available surface area is decreased. Studies performed with bystander red cell-bound C3d demonstrated in vivo complement activation occurring in six patients receiving Fluosol as an adjunct to chemotherapy for colon cancer. In two patients, there was a marked increase in red cell-bound C3d after Fluosol infusion; these two patients also developed adverse reactions during Fluosol infusion. These studies suggest that the Fluosol surface plays a major role in the initiation and regulation of complement activation that is seen during Fluosol infusion.     

EASE OF HANDLING AND CLINICAL EFFICACY OF FLUTICASONE PROPIONATE ACCUHALER/DISKUS™ INHALER COMPARED WITH THE TURBOHALER™ INHALER IN PAEDIATRIC PATIENTS

A total of 323 children aged 4-11 years who were receiving, or had symptoms indicating a clinical requirement for, inhaled corticosteroid at a daily dose of 400 μg budesonide (BUD) or beclomethasone dipropionate (BDP), or 200 μg fluticasone propionate (FP), were randomised into this multicentre, open-label, parallel group study. Patients received either FP 100 μg b.d. administered via the Accuhaler/Diskus inhaler (n=159) or BUD 200 μg b.d. administered via a Turbohaler inhaler (n=164) for four weeks and recorded daily their morning and evening peak expiratory flow (PEF), asthma symptoms and use of relief medication. Device handling was assessed by a questionnaire, with responses recorded on three- or five-point ordinal scales. The primary efficacy parameter was mean percent predicted morning PEF. The device handling results showed the Accuhaler/Diskus inhaler was rated more favourably than the Turbohaler inhaler in terms of ease of correct inhaler use, ease of telling how many doses were left, ease of knowing whether a dose had been inhaled and overall liking of the device. More patients in the Accuhaler/Diskus group (85%) than in the Turbohaler group (58%) said they would be happy to receive the same device again, while 8% and 25% respectively said they would not be happy to be given it again. In addition, the change from baseline to week 4 of treatment in mean percent predicted morning PEF was greater in the FP Accuhaler/Diskus group, indicating that FP 200 μg daily via Accuhaler/Diskus inhaler is at least as clinically effective as BUD 400 μg daily via the Turbohaler inhaler.     

Multiple granular cell tumors are an associated feature of LEOPARD syndrome caused by mutation in PTPN11

We report a patient with a clinical and molecular diagnosis of LEOPARD syndrome (LS) associated with multiple granular cell tumors (MGCT). Bidirectional sequencing of exons 7, 12, and 13 of the PTPN11 gene revealed the T468M missense mutation in exon 12. This mutation has been previously reported in patients with LS. To our knowledge, this is the first report of MGCT associated with molecularly characterized LS and provides the first molecular evidence linking granular cell tumors (GCT) to the Ras/mitogen-activated protein (MAP) kinase pathway. We propose that MGCT can be associated with LS. Analysis of GCT from this case tested negatively for loss of heterozygosity (LOH) at the PTPN11 and NF1 loci and did not show deletions of the PTEN gene. The absence of LOH of PTPN11 supports published functional data that T468M is a dominant-negative mutation.     

The factor V B-domain provides two functions to facilitate thrombin cleavage and release of the light chain

Blood coagulation factors V and VIII are homologous proteins that have the domain organization A1-A2-B-A3-C1-C2. Upon thrombin activation, the B-domains of both molecules are released. Previous studies on factor VIII showed that the B-domain was not required for thrombin cleavage or activity. In contrast, deletion of the factor V B-domain (residues 709 to 1545) yielded a molecule with sevenfold reduced procoagulant activity that was not cleaved by thrombin. However, this factor V B- domain deletion molecule was activated by factor Xa, although the fold- activation was 85% that of wild-type factor V. Thrombin cleavage of factor V occurs initially after residue 709 and subsequently after residues 1018 and 1545. The requirement for thrombin cleavage within the B-domain at residue 1018 was evaluated by mutagenesis of Arg1018 to Ile. In the resultant R1018I mutant, the rate of thrombin activation and appearance of maximal cofactor activity was delayed and was consistent with delayed cleavage of the light chain at residue 1545. In contrast, the rate of factor Xa activation in the R1018I mutant was not altered. This finding suggests that thrombin cleavage at 1018 facilitates subsequent thrombin cleavage at 1545. Further mutagenesis was used to study the requirement for sequences within the factor V B- domain for thrombin cleavage at residue 1545. Whereas the factor V deletion molecule removing residues 709 to 1545 was not cleaved by thrombin, a smaller B-domain deletion molecule (residues 709 to 1476) containing an acidic amino acid-rich region (residues 1490 to 1520) was effectively cleaved by thrombin. These results show that residues 1476 to 1545, which contain an acidic amino acid-rich region, were required for thrombin cleavage of the light chain. Introduction of an acidic amino acid-rich region from factor VIII (residues 337 to 372) into the factor V 709 to 1545 deletion also restored thrombin cleavage of the light chain. In contrast, similar replacement with the acidic region from the factor VIII light chain (residues 1649 to 1689) was significantly less effective in promoting thrombin cleavage of the light chain. This finding suggests that the different acidic regions in factors V and VIII are not functionally equivalent in their interaction with thrombin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Predictors of successful labor induction with oral or vaginal misoprostol

Objective: To identify independent predictors of successful labor induction with oral or vaginal misoprostol.

Methods: Women enrolled in four previous randomized trials involving oral or vaginal misoprostol for cervical ripening and labor induction were included in the present cohort study, with dosing of 25–50?μg every 4 to 6?h vaginally (n?=?574) or 50?μg every 4?h orally (n?=?207). Multiple logistic regression was performed to identify factors independently associated with successful labor induction – defined as vaginal delivery within 12?h, vaginal delivery within 24?h and spontaneous vaginal delivery. Predictors of Cesarean birth and the need for only one dose of misoprostol were also identified. Variables included in the models were maternal age, weight, height, parity, gravidity, membrane status, route of misoprostol, gestational age, birth weight, and Bishop score and its individual components.

Results: Maternal age, height, weight, parity, birth weight, dilatation, effacement and cervical station were associated with vaginal delivery within 24?h of induction. Maternal age, height, weight, nulliparity, birth weight and route of misoprostol were associated with Cesarean birth, with oral misoprostol being associated with a lower rate of Cesarean birth. The need for only one dose of misoprostol was predicted by maternal height, weight, parity, gestational age, Bishop score and route of misoprostol.

Conclusion: Characteristics of the woman (height, weight, parity), the fetus (birth weight) and some of the individual components of the Bishop score, were associated with successful labor induction, with oral misoprostol being associated with a lower rate of Cesarean birth.     

Molecular characterization of human factor XSan Antonio

Enzymatic amplification technique was used to isolate all eight exons and sequences around the splice junctions, putative promoter, and polyadenylation sites of human factor X DNA from a patient with factor X deficiency. Two genetic changes in factor X have been observed in this patient. The patient is most likely a compound heterozygote since there is only 14% activity associated with factor X. A point mutation that resulted in the substitution of cysteine (TGC) for arginine (CGC) at amino acid 366 was found in exon VIII of one allele of the factor X gene. This mutation, which occurs in the catalytic domain, can affect the formation of a disulfide bridge and thus could result in a reduction in factor X activity. Sequencing all the regions revealed a second mutation: a deletion of one nucleotide (TCCT to TCT) in exon VII that would cause a frame shift at amino acid 272 followed by termination. We have also shown that the point mutation in exon VIII creates an ApaL1 restriction site and destroys the HinP1 site. Enzymatic DNA amplification followed by restriction digestion provides a quick, reliable, and sensitive method for carrier detection and antenatal diagnosis in affected kindreds. This is the first characterization of factor X deficiency at the molecular level. We propose to name this mutation Factor XSan Antonio.     

Effect of adrenomedullin on the cerebral circulation: relevance to primary headache disorders

Adrenomedullin (ADM) is closely related to calcitonin gene-related peptide, which has a known causative role in migraine. Animal studies have strongly suggested that ADM has a vasodilatory effect within the cerebral circulation. For these reasons, ADM is also likely to be involved in migraine. However, the hypothetical migraine-inducing property and effect on human cerebral circulation of ADM have not previously been investigated. Human ADM (0.08 µg kg⁻¹ min⁻¹) or placebo (saline 0.9%) was administered as a 20-min intravenous infusion to 12 patients suffering from migraine without aura in a crossover double-blind study. The occurrence of headache and associated symptoms were registered regularly 24 h post infusion. Cerebral blood flow (CBF) was measured by ¹³³Xenon single-photon emission computed tomography, mean blood flow velocity in the middle cerebral artery (V_MCA) by transcranial Doppler and the diameter of peripheral arteries by transdermal ultrasound (C-scan). ADM did not induce significantly more headache or migraine compared with placebo ( P  = 0.58). CBF was unaffected by ADM infusion (global CBF, P  = 0.32 and rCBF_MCA, P  = 0.38) and the same applied for the V_MCA ( P  = 0.18). The superficial temporal artery dilated compared with placebo ( P  < 0.001), and facial flushing was seen after ADM administration ( P  = 0.001). In conclusion, intravenous ADM is not a mediator of migraine headache and does not dilate intracranial arteries.