The P303T mutation in the human factor VII (FVII) gene alters the conformational state of the enzyme and causes a severe functional deficiency

We report the results of in vitro expression and biochemical characterization of the naturally occurring type II mutation Pro303Thr (P303T) in the factor VII (FVII) gene. Recombinant activated mutated FVII (FVIIa303T), compared with the activated wild-type FVII (FVIIaWT), showed reduced amidase activity toward synthetic substrates, especially when the observed reduced binding affinity for human soluble tissue factor (TF) (K(d) from 4.4 nmol/l for FVIIaWT to 17.3 nmol/l for FVIIa303T) was overcome by a fully saturating TF concentration. Likewise, factor X (FX) hydrolysis by FVIIa303T showed a reduced activity in the absence (and more severely in the presence) of TF (k(cat)/K(m) from 2.3 x 10(7)/mol/l s for FVIIaWT to 8.7 x 10(5)/mol/l s for FVIIa303T). These results showed that the mutant FVIIa is more shifted toward a zymogen-like form compared to FVIIaWT, suggesting that P303 facilitates the conformational transitions that stabilize the active form of FVIIa. The alteration of these allosteric equilibria is especially evident in the presence of TF, which was unable to shift the equilibrium toward a fully active FVIIa form. Additional experiments showed that both TF-catalysed FVII303T autoactivation and FVII303T activation by activated FX in the presence of TF were severely impaired, mainly because of an increase of the K(m) value. Altogether, these defects may explain the severe bleeding symptoms in a patient carrying the FVIIP303T mutation.     

TNFRSF11B gene polymorphisms increased risk of peripheral arterial occlusive disease and critical limb ischemia in patients with type 2 diabetes

Aims

Osteoprotegerin (OPG) is a secretory glycoprotein that belongs to the tumor necrosis factor receptor family and plays a role in atherosclerosis. OPG has been hypothesized to modulate vascular functions; however, its role in mediating atherosclerosis is controversial. Epidemiological data in patients with cardiovascular disease (CVD) indicate that OPG serum levels are associated with several inflammatory markers, myocardial infarction events, and calcium scores, suggesting that OPG may be causative for CVD.

Methods

The present study aimed to evaluate whether the OPG gene (TNFRSF11B) polymorphisms are involved in the development of peripheral arterial occlusive disease (PAOD) and critical limb ischemia (CLI) in patients with type 2 diabetes. This genetic association study included 402 diabetic patients (139 males and 263 females) with peripheral arterial occlusive disease and 567 diabetic subjects without peripheral arterial occlusive disease (208 males and 359 females). The T245G, T950C, and G1181C polymorphisms of the OPG gene were analyzed by polymerase chain reaction and restriction fragment length polymorphism.

Results

We found that the T245G, T950C, and G1181C gene polymorphisms of the OPG gene were significantly (27.9 vs. 12.2 %, P < 0.01; 33.6 vs. 10.4 %, P < 0.01 and 24.4 vs. 12.7 %, P < 0.01, respectively) and independently (adjusted OR 4.97 (3.12–6.91), OR 7.02 (4.96–11.67), and OR 2.85 (1.95–4.02), respectively) associated with PAOD. We also found that these three polymorphisms act synergistically in patients with PAOD and are associated with different levels of risk for PAOD and CLI, depending on the number of high-risk genotypes carried concomitantly by a given individual.

Conclusion

The TNFRSF11B gene polymorphisms under study are associated with PAOD, and synergistic effects between these genotypes might be potential markers for the presence and severity of atherosclerotic disorders.     

Immunogenicity and safety of Fluzone intradermal and high-dose influenza vaccines in older adults ≥65 years of age: A randomized,controlled, phase II trial

We conducted a randomized, controlled, multicenter, phase II study to evaluate the immunogenicity and safety of an investigational intradermal (ID) trivalent influenza vaccine (TIV) and a high-dose (HD) intramuscular (IM) TIV in older adults (≥65 years of age). Older adult subjects were immunized with ID vaccine containing either 15 μg hemagglutinin (HA)/strain (n = 636) or 21 μg HA/strain (n = 634), with HD IM vaccine containing 60 μg HA/strain (n = 320), or with standard-dose (SD) IM vaccine (Fluzone^®; 15 μg HA/strain; n = 319). For comparison, younger adults (18–49 years of age) were immunized with SD IM vaccine. In older adults, post-vaccination geometric mean titers induced by the ID vaccines were superior to those induced by the SD IM vaccine for the A/H1N1 and A/H3N2 strains and non-inferior for the B strain. Seroconversion rates induced by the ID vaccines were superior to those induced by the SD IM vaccine in older adults for the A/H1N1 and B strains and non-inferior for the A/H3N2 strain. Results did not differ significantly for the two ID vaccine dosages. Post-vaccination geometric mean titers, seroconversion rates, and most seroprotection rates were significantly higher in HD vaccine recipients than in older adult recipients of the SD IM or ID vaccines and, for most measures, were comparable to those of younger adult SD IM vaccine recipients. Injection-site reactions, but not systemic reactions or unsolicited adverse events, were more common with the ID vaccines than with the IM vaccines. No treatment-related serious adverse events were reported. This study demonstrated that: (1) the ID and HD vaccines were well-tolerated and more immunogenic than the SD IM vaccine in older adults; (2) the HD vaccine was more immunogenic than the ID vaccines in older adults; and (3) the HD vaccine in older adults and the SD IM vaccine in younger adults elicited comparable antibody responses (ClinicalTrials.gov identifier no.: NCT00551031).     

Macular holes

Spontaneous closure of macular holes occurs in 3% to 9% of patients over 6 years [3,58]. Visual acuity can recover dramatically once the hole closes. The risk for hole development in the fellow eye in the absence of PVD is approximately 29%. The presence of vitreomacular separation reduces the risk to less than 5%. Most of the fellow eyes that develop macular holes do so within 2 years [59]. Macular hole surgery has been evolving. ILM peeling is a recent, widely accepted innovation. Meta-analysis of 12 published case series indicates anatomic success in 77% and functional success in 55% of patients with the traditional technique of epiretinal peeling and no adjuvant use. Meta-analysis of 22 series with techniques using adjuvants indicates an anatomic success rate of 81% and a functional success rate of 60%. Meta-analysis of 4 studies involving 221 cases indicates an anatomic success rate of 96% and a functional success rate of 81% [5]. True superiority of one approach versus another cannot be determined without a randomized, prospective clinical trial, which is unlikely to be conducted. In the meantime, surgeons must choose an approach based on individual features of a given patient and their own surgical experience.     

Plasma protein oxidation is associated with an increase of procoagulant markers causing an imbalance between pro- and anticoagulant pathways in healthy subjects

The aim of the present study was to investigate whether the overall oxidation state of plasma proteins is associated with changes of circulating pro- and anticoagulant markers in healthy subjects (n = 99, 49 males, 50 females, aged from 6 to 91 yrs.). The carbonyl content of plasma proteins was measured and validated as an ex vivo index of the overall protein oxidation state due to its correlation with the plasma level of o-tyrosine (r = 0.87, P <0.0001), which is a well known oxidized product of L-phenylalanine. Using a multivariate analysis the carbonyl content of plasma protein was positively associated with procoagulant markers such as prothrombin F1 + 2 (r = 0.28, P = 0.0019) and fibrinopeptide A, (FpA) (r = 0.278, P = 0.003), as well as with the soluble derivative of the endothelial protein thrombomodulin (TM) (r = 0.469, P <0.0001). The procoagulant marker of thrombin activity, FpA, was significantly and positively correlated with the anticoagulant product of thrombin, namely the Protein C activation peptide (PCP), only in the tertile with low protein carbonyl content. At higher tertiles this correlation was no longer observed, thus suggesting a detrimental effect of oxidative stress on the TM/Protein C anticoagulant pathway. In 15 subjects with high carbonyl content of plasma protein, treatment for 18 days with 600 mg/d of vitamin E did not substantially modify the protein carbonyl content, the anticoagulant markers APC/PCP, and all procoagulant markers except F1+2, whose value significantly decreased by 25%. In conclusion, the present study shows that a high plasma protein oxidation ex vivo is associated with an overall hemostatic imbalance, which favors procoagulant markers. Vitamin E treatment in vivo restores only in part the equilibrium between pro- and anticoagulant pathways. This may open the way to further studies aimed at elucidating the mechanisms by which the oxidative stress is linked to activation of the coagulation system in atherothrombotic disorders.     

Oxidation of human alpha-thrombin by the myeloperoxidase-H2O2-chloride system: structural and functional effects

The myeloperoxidase-H2O2-chloride system (MPOS) is exploited by white blood cells to generate reactive oxygen species in many processes involved in the pathogenesis of inflammation and atherothrombosis. This, study investigated the biochemical and functional effects of alpha-thrombin oxidation by MPOS. This system, in the presence of 100 microM L-tyrosine, caused in the thrombin molecule loss of tryptophan and lysine residues and formation of dityrosine, chloramine and carbonyl groups. The same changes could be directly induced by thrombin incubation with reagent HOCI, but not with H2O2 alone. Exposure to either MPOS or HOCl caused major functional abnormalities in human alpha-thrombin. The interaction of oxidized (ox-)thrombin with Protein C and antithrombin III-heparin complex were most sensitive to oxidation, being the kcat/Km value for Protein C hydrolysis roughly reduced 13-fold and the affinity for the antithrombin III-heparin complex decreased approximately 15-fold. Ox-thrombin interaction with small synthetic peptides showed several changes, arising from a perturbation of the S2-S3 specificity of the enzyme. Ox-thrombin was also characterized by a 5-fold decrease of the kcat/Km value for both fibrinopeptide A and B release from fibrinogen, a 5.8-fold increase of the EC50 value for platelet activation and a 2-fold decrease of binding affinity for thrombomodulin. The above results indicate a high sensitivity of thrombin to oxidative modifications by myeloperoxidase. Perturbed interactions with Protein C and the heparin-ATIII complex were the most relevant functional abnormalities of ox-thrombin.     

Abnormal pH-sensing of platelet Na+/H+ exchanger in patients with cardiac syndrome X

An enhanced activity of Na⁺/Li⁺ countertransport, studied as a surrogate of Na⁺/H⁺ exchanger, has been described in red blood cells of patients with cardiac syndrome X. In this study we investigated whether abnormalities in the activity of platelet Na⁺/H⁺ exchanger (NHE) also existed in syndrome X patients and whether such abnormality was associated with platelet activation. Platelet NHE activity was evaluated in 21 syndrome X patients and 18 controls by measuring the pH recovery in platelets after acid loading and/or thrombin stimulation. The linear correlation existing between the initial intracytoplasmic pH (pH_i) values and the maximal velocity of pH recovery allowed to calculate the values of slope and intercept at pH_i=6.6 (I_pH6.6) for each individual. Urinary excretion of the major TXB₂ metabolite, 11-dehydro-TXB₂ was measured in 15 syndrome X patients and 15 controls. The acidification-induced NHE activity resulted significantly higher in syndrome X patients compared to controls. Indeed, slope values were 0.75±0.29 and 0.5±0.23 min⁻¹ in patients and controls, respectively (P=0.01), while I_pH6.6 values were 0.24±0.1 and 0.17±0.1 ΔpH/min (P=0.04). The thrombin-stimulated NHE activity, however, was not different in the two groups and no significant difference in the urinary excretion of 11-dehydro-TXB₂ between patients and controls (median 920 vs. 765 pg/mg creatinine, respectively) (P=0.32) was also found. Thus our data demonstrate an alkaline shift in pH-dependence of platelet NHE of syndrome X patients. This abnormality does not seem to be associated with increased platelet activation.     

Paraneoplastic limbic encephalitis and possible narcolepsy in a patient with testicular cancer: case study

We describe a patient who presented with a clinical syndrome of limbic encephalitis, narcolepsy, and cataplexy. The anti-Ma2 antibody was positive. Although there was no mass on imaging, orchiectomy was performed in this patient, and testicular carcinoma was found. This is the first known case of limbic encephalitis and anti-Ma2 antibody to be associated with cataplexy and possible narcolepsy. Neurological symptoms precede the diagnosis of cancer in 50% of patients with paraneoplastic syndromes, and clinicians are therefore strongly advised to evaluate patients with neurological symptoms for this condition.     

Fipexide improvement of cognitive functions in rat: behavioural and neurochemical studies

Dopamine neurotransmission plays a role in learning, memory and related cognitive processes. We evaluated the effect of a nootropic drug, fipexide, a parachloro-phenossiacetic acid derivative, both on behavioural and biochemical parameters. The compound was able to cause an improved performance on active avoidance test, when administered just before the trial, being also effective on memory retention and recall experiments. Fipexide was also able to revert the impairment of the acquired behaviour caused by sulpiride in pretrained rats, while it failed to be effective on the haloperidol deconditioning effect. Striatal adenylate cyclase activity, from fipexide pretreated rats, was significantly reduced both in basal and dopamine stimulated conditions. Furthermore, fipexide, directly added to membrane preparations, was able to inhibit striatal adenylate cyclase activity. These results, along with our previous reports on fipexide effect on locomotor activity, allow us to hypothesize that the positive effect of this drug on cognitive performance seems to be mediated, at least partially, by dopaminergic neurotransmission.     

Combination of 6-mercaptopurine, vincristine, methotrexate, and prednisone in resistant or relapsed acute nonlymphoid leukemia

Twenty-six adults with acute nonlymphoid leukemia (ANLL) relapsed or resistant after first-choice chemotherapy including cytarabine and/or anthracyclines were treated with a modified POMP (6-mercaptopurine, vincristine, methotrexate, and prednisone) combination. Complete remission (CR) was achieved in five of eight resistant patients (62%) and in nine of 18 relapsed patients (50%). Intermittent cycles of chemotherapy including daunorubicin, cytarabine, 6-thioguanine, and etoposide were then administered. In the resistant group CR lasted from 11 to 126 weeks (median, 86), with two patients remaining in CR 126 and 108 weeks after remission induction; in relapsed patients, CR lasted from 9 to 50 weeks (median, 31).