Efficacy and safety during formulation switch of a pasteurized VWF/FVIII concentrate: results from an Italian prospective observational study in patients with von Willebrand disease.

Von Willebrand disease (VWD) is an inherited bleeding disorder caused by the quantitative or qualitative deficiency of von Willebrand factor (VWF). Replacement therapy with plasma-derived VWF/factor VIII (FVIII) concentrates is required in patients unresponsive to desmopressin. To assess the efficacy, safety and ease of use of a new, volume-reduced (VR) formulation of VWF/FVIII concentrate Haemate(®) P in patients requiring treatment for bleeding or prophylaxis for recurrent bleeding or for invasive procedures. Pharmacoeconomic variables were also recorded. Data were analysed using descriptive statistics. This was a multicentre, prospective, observational study. Consecutively enrolled patients received Haemate(®) P VR according to their needs, and were followed for 24 months. Of the 121 patients enrolled, 25.6% had type 3 VWD and more than 40% had severe disease. All patients were followed for 2 years, for a total of 521 visits. On-demand treatment was given to 61.9% of patients, secondary long-term prophylaxis to 25.6% and prophylaxis for surgery, dental or invasive procedures to 45.5%. The response to treatment was rated as good to excellent in >93-99% of interventions. The new formulation was well tolerated by all patients with no report of drug-related adverse events. The switch to volume-reduced Haemate(®) P was easy to perform and infusion duration was decreased twofold compared with the previous formulation. Volume-reduced Haemate(®) P was at least as effective and well-tolerated as the previous formulation.

Intraoperative intravenous administration of rFVIIa and hematoma volume after early surgery for spontaneous intracerebral hemorrhage: a randomized prospective phase II study.

BACKGROUND: Surgery of spontaneous supratentorial intracerebral hemorrhage (ICH), especially if performed early, can be complicated by rebleeding, a condition that can worsen the outcome. We evaluated the effect of recombinant activated factor VII (rFVIIa) on postoperative rebleeding. METHODS: In this randomized, open-label, single-blinded study, 21 patients with spontaneous supratentorial ICH diagnosed by computed tomography (CT) scan were treated with intravenous rFVIIa (100 mcg/Kg b.w., N=13) or placebo (N=8). Hematoma volume was assessed using CT scan immediately, 18-30 hours, and 5-7 days after hematoma evacuation. The primary endpoint was a hematoma volume at 18-30 hours after surgery. All CT scans were evaluated at one center by the same investigator who was unaware of the treatment. Hematoma volume was measured using dedicated software. RESULTS: At baseline, the hematoma volume was 59.2±27.4 and 71.5±32.1 mL in the rFVIIa and placebo group, respectively. Hematoma evacuation resulted in significantly smaller ICH volumes that were similar in the rFVIIa and placebo group at 18-30 hours after surgery (15.9±14.2 mL and 18±15.1 mL, respectively; mean difference 2.1 mL, 95% confidence interval -12.1 to 16.2, P=0.76 (0.03 mL after adjustment for baseline value)). The frequencies of deep venous thrombosis, myocardial infarction, troponin I elevation and cerebral ischemia were similar in both groups. CONCLUSION: In this pilot study, intraoperative, intravenous rFVIIa administration did not modify hematoma volume after early ICH surgery. However, the 95% CI was wide, which indicates considerable uncertainty. Therefore, our results do not disprove the potential benefit of rFVIIa administration, which could be shown in a larger study.

Thrombophilic screening in Turner syndrome.

AIM: The aim of this study was to determine, in patients with Turner syndrome (TS), the prevalence of thrombophilic disorders correlating with a higher risk of venous thromboembolism (VTE), to evaluate if thrombophilia is associated with the genetic features of these patients and whether screening before hormone replacement therapy (HRT) is advisable. PATIENTS AND METHODS: We examined 82 TS patients. In all patients we analyzed activated factor VIII:C, fibrinogen, antithrombin (AT), protein C (PC), protein S (PS), activated PC resistance, and homocysteine. For every patient, an investigation for mutations in prothrombin G20210A, factor V R506Q, methylenetetrahydropholate reductase (MTHFR) C 677T and A1298C was conducted. RESULTS: Low values of PC in 3 patients (3.70%), low values of PS in 12 (14.81%), and hyperhomocysteinemia in 4 (4.87%) were found; 52 girls (64.2%) presented hyperfibrinogenemia. Three patients were heterozygous for the prothrombin G20210A allele mutation (3.66%) and the factor V mutation was present in 4 patients (4.88%). No TS patient had a homozygous mutation. Mutations in the MTHFR gene were present in 62 girls, in 17 patients (20.7%) they were homozygous and in 45 patients (54.88%) heterozygous. CONCLUSIONS: Considering the increased risks with the association between VTE and the higher prevalence of PC and PS deficiencies, TT genotype mutations and high level of fibrinogen, it is advisable to perform a complete thrombophilia screening in TS patients before starting HRT.

Homocysteine, vitamin B12 and folic acid levels in psoriatic patients and correlation with disease severity.

Hyperhomocysteinaemia represents an independent risk factor for atherosclerotic cardiovascular disease, stroke, peripheral arterial occlusive disease and venous thrombosis. Psoriasis is a chronic inflammatory skin disease associated with increased atherothrombosis and cardiovascular risk profile. The aim of this study is to investigate homocysteine, folic acid and vitamin B12 levels in a cohort of psoriatic patients and its relationship with the severity of the disease. A retrospective observational study in 98 patients with chronic plaque psoriasis and 98 healthy controls was performed. Total plasma homocysteine level, folic acid, vitamin B12 and PASI index were assessed in every patient. Patients with psoriasis had plasma homocysteine levels higher than controls (57% of cases and 25% of controls; p<0.0001). Folic acid and vitamin B12 plasma levels were lower in psoriatic patients than in controls (p = NS), lower levels of vitamin B12 were found in patients with hyperhomocysteinaemia compared to patients with a normal value of homocysteine (p = 0.0009). The severity of psoriasis assessed according to PASI (19.51+/-16.26) did not directly correlate either with higher levels of homocysteine or with vitamin B12 and folic acid plasma levels. In conclusion, a significantly higher prevalence of hyperhomocysteinaemia was found in psoriatic patients compared to healthy controls. A significant correlation between hyperhomocysteinaemia and lower vitamin B12 levels, but not folic acid, was evidenced. On the contrary, our data do not correlate the high level of homocysteine with higher PASI scores or psoriasis type, suggesting that homocysteine level can be considered an independent risk factor in psoriatic patients.

Mitral valve prolapse and abnormalities of haemostasis in children and adolescents with migraine with aura and other idiopathic headaches: a pilot study.

OBJECTIVE: To investigate the prevalence of mitral valve prolapse (MVP) and abnormalities of haemostasis in children and adolescents with migraine with aura (MA) compared with peers affected by other idiopathic headaches. MATERIALS AND METHODS: We recruited 20 MA patients (10 men and 10 women; age range 8-17 years) and 20 sex- and age-matched subjects with other idiopathic headaches. Both groups underwent colour Doppler transthoracic echocardiography to detect MVP and the following laboratory work-up: plasma prothrombin time, activated partial thromboplastin time, thrombin time, fibrinogen, protein C, protein S, homocysteine, lupus anticoagulant, von Willebrand factor (vWF) ristocetin cofactor activity, immunoglobulins (Ig) G and M anticardiolipin antibodies (aCL). Factor V Leiden, factor II and methylenetetrahydrofolate reductase were investigated (we did not test the entire genes, but screened for specific point mutations). RESULTS: The prevalence of MVP was significantly higher in the MA subjects than in the patients affected by other idiopathic headaches (40% vs 10%; P < 0.05). Moreover, the MA patients showed a higher rate of above-normal IgM aCL titres (45% vs 10%; P < 0.05). Finally, in the group of patients with MVP we found a higher prevalence of aCL in those with MA compared with those affected by other idiopathic headaches. CONCLUSIONS: A proportion, at least, of the MA patients showed a more complex phenotype characterized by MVP and/or positive aCL titres. The pathogenetic role of these associations is obscure and larger studies are needed to confirm the usefulness of echocardiographic and laboratory investigations in this area and to identify possible new treatment approaches that might be explored in this group of MA patients.

Platelet-Independent defect in hemostasis associated with sirolimus use.

Sirolimus is currently used to prevent rejection of solid organ transplant, and sirolimus-eluting stents have shown promise for the prevention of coronary artery restenosis. Thrombocytopenia is a well-known adverse effect of sirolimus limiting its use. Herein we report on a patient in whom sirolimus caused a platelet-independent hemostasis defect. The patient was a 52-year-old woman who underwent renal transplant with consequent normal kidney function. The immunosuppressive regimen included basiliximab, steroids, and cyclosporine induction later shifted to sirolimus and mycophenolate due to biopsy findings of tubular necrosis on day 6 posttransplantation. At discharge the serum creatinine was 0.7 mg/dL. Four months after transplantation the patient was admitted to our hospital because of fever (37.5 degrees C to 38 degrees C), anorexia, and asthenia. Blood analysis showed: creatinine 1.7 mg/dL, Hb 9.6 g/dL, WBC 6 x 10(3)/microL, PLT 123 x 10(3)/microL, liver function tests normal, LDH 720 mU/mL, fibrinogen 628 mg/dL, d-dimer 0.42 ng/mL, FDP > 40 ng/mL, INR 1.10, PT 87%, aPTT 40 seconds. Cultures and tests for infection were negative. Serum sirolimus level was 25.9 ng/mL. The following day the serum creatinine rose to 2.3 mg/dL and diuresis fell to 20 mL/h. Multiple bleeding times (Ivy test) performed before the renal biopsy were repeatedly over 30 minutes (normal 3 to 5 minutes), despite normal platelet count and platelet function studies. There was no spontaneous aggregation and in vitro aggregation was normal (collagen, ADP, adrenalin, and ristocetin induced). Coagulation studies showed a defect in fibrin formation and a reduction of fibrinolysis. Suspension of sirolimus treatment was followed by remission of fever, improvement of renal function (serum creatinine 1.2 mg/dL), and normalization of bleeding time.

Alternative splicing and diversity of renal transporters.

The growing molecular identification of renal transporter genes is revealing that alternative splicing is common among transporters. In this paper, I review the physiological consequences of alternative splicing in some genes encoding renal transporters in which spliced isoforms have recently been identified. In some cases, the spliced isoforms resulted in nonfunctional proteins, which, however, possess a dominant negative effect on the cotransporter function, suggesting that the presence of such isoforms can be important in the functional regulation of the transporter. In most transporter genes, however, the spliced isoforms have been shown to be functional, resulting in a variety of physiological consequences, including, for example, changes in the polarization of isoforms to the apical or basolateral membrane, changes in pharmacological or kinetic properties, and changes in tissue distribution or intrarenal localization. In some cases, although the spliced isoform is functional, the consequence of splicing is still unknown. Different regulation among isoforms is an interesting possibility. Thus the diversity of several renal transporters is enhanced by alternative splicing mechanisms.

Renal potassium-chloride cotransporters.

The electroneutral cotransport of potassium and chloride is mediated by potassium-chloride transporters, which are encoded by members of the gene family of cation-chloride cotransporters. A significant body of evidence argues for swelling-activated, basolateral potassium-chloride transport in the proximal tubule and thick ascending limb, with a potential role in transepithelial salt transport. However, the lack of specific inhibitors has impeded progress in this area. The cloning of the four potassium-chloride cotransporter genes has sparked new interest in this transport pathway, and promises to yield novel insights into their roles in cellular and renal physiology.

Safety and efficacy of thalidomide in patients with myelofibrosis with myeloid metaplasia.

We administered the anti-angiogenic drug thalidomide to 21 patients (12 men) with myelofibrosis with myeloid metaplasia (MMM), who were not responsive to standard treatment. Patients received thalidomide at an escalating dose from 100 to 400 mg/d. Administration of the drug was discontinued before the planned 6 months of treatment in 19 patients (90.5%), mainly because of somnolence and/or fatigue, neurological symptoms or neutropenia. Of the 13 evaluable patients (who received more than 30 d of therapy), anaemia improved in three out of seven (43%) who were treated because of anaemia; thrombocytopenia improved in two out of three (66.6%) who were treated because of thrombocytopenia; splenomegaly was reduced in four (30.8%). Undesired increases in white blood cell and platelet counts were observed in three (23.1%) and five (38.5%) patients respectively. A severity score, indexed on haematological and clinical parameters, improved in two patients (15.4%), but worsened in five (38.5%). In conclusion, standard-dose thalidomide in MMM patients is burdened with a high rate of side-effects, which prevent prolonged treatment. Because the drug is effective in improving anaemia and thrombocytopenia and in reducing splenomegaly, low-dose therapy warrants evaluation. The unexpected observation of leucocytosis and thrombocytosis suggests biological studies and better criteria for selection of patients for treatment.

Functional and molecular characterization of the K-Cl cotransporter of Xenopus laevis oocytes.

The K-Cl cotransporters (KCCs) have a broad range of physiological roles, in a number of cells and species. We report here that Xenopus laevis oocytes express a K-Cl cotransporter with significant functional and molecular similarity to mammalian KCCs. Under isotonic conditions, defolliculated oocytes exhibit a Cl(-)-dependent (86)Rb(+) uptake mechanism after activation by the cysteine-reactive compounds N-ethylmaleimide (NEM) and mercuric chloride (HgCl(2)). The activation of this K-Cl cotransporter by cell swelling is prevented by inhibition of protein phosphatase-1 with calyculin A; NEM activation of the transporter was not blocked by phosphatase inhibition. Kinetic characterization reveals apparent values for the Michaelis-Menten constant of 27.7 +/- 3.0 and 15.4 +/- 4.7 mM for Rb(+) and Cl(-), respectively, with an anion selectivity for K(+) transport of Cl(-) = PO(4)(3-) = Br(-) > I(-) > SCN(-) > gluconate. The oocyte K-Cl cotransporter was sensitive to several inhibitors, including loop diuretics, with apparent half-maximal inhibition values of 200 and 500 microM for furosemide and bumetanide, respectively. A partial cDNA encoding the Xenopus K-Cl cotransporter was cloned from oocyte RNA; the corresponding transcript is widely expressed in Xenopus tissues. The predicted COOH-terminal protein fragment exhibited particular homology to the KCC1/KCC3 subgroup of the mammalian KCCs, and the functional characteristics are the most similar to those of KCC1 (Mercado A, Song L, Vazquez N, Mount DB, and Gamba G. J Biol Chem 275: 30326--30334, 2000).

Peritoneal protein loss in patients with high peritoneal permeability: comparison between continuous ambulatory peritoneal dialysis and daytime intermittent peritoneal dialysis.

BACKGROUND: Dialysate protein loss is involved in the etiology of hypoalbuminemia and malnutrition on continuous ambulatory peritoneal dialysis (CAPD). Patients with high peritoneal membrane permeability had the lowest serum albumin (Alb) and highest dialysate protein concentrations and achieved higher small solute dialysis/plasma equilibration in a shorter time than patients with low peritoneal transport. The aim of this prospective crossover study was to evaluate whether protein loss might be decreased in patients with high peritoneal permeability on short dwell-time (DT) peritoneal dialysis. METHODS: Five high and nine high-average peritoneal transport patients were subjected to the following sequential dialysis schemes (four exchanges/day, glucose 1.5%): scheme A, three daytime exchanges (4-6 h DT) and one nightly (8-12 h DT) for 2-3 days, scheme B, 3-h DT each and dry peritoneum at night during 5 days, a wash-out period similar to scheme A, and scheme C, 2-h DT each and dry peritoneum the remainder of day and night during 5 days. Dialysate Alb, IgG, IgA, and IgM losses and adequacy of dialysis were evaluated at the end of each scheme. RESULTS: Dialysate IgM was not detected. All protein losses were reduced with the short DT dialysis schemes; however, dialysis CCl and KT/V(urea) were also decreased. In patients with high peritoneal transport type, the 3-h DT dialysis scheme achieved a reduction in Alb loss without significant reduction of adequacy of dialysis. CONCLUSIONS: Peritoneal Alb, IgG, and IgA losses are significantly reduced in patients with high peritoneal permeability on short dwell-time dialysis and extended dry periods. However, a reduction of dialysis contribution to small solute clearances was also observed, Three-hour dwell-time dialysis may be particularly useful in patients with high peritoneal transport type, as it tends to reduce peritoneal protein loss without notably reducing adequacy of dialysis.

[Viral hepatitis C in patients with terminal chronic renal insufficiency. III. Viral quantification]. / Hepatitis viral C en pacientes con insuficiencia renal crónica terminal. III. Cuantificación viral.

BACKGROUND: We have previously shown that the prevalence of hepatitis associated with the hepatitis C virus (HCV) in patients with end stage renal disease in our institution is 10.2%. However, quantification of viral RNA in plasma and its relation with clinical variables has never been studied in our patients. Thus, the aim of the present work was to quantify the HCV viral load in patients with ESRD in dialysis, and to correlate these values with the dialysis modality and the viral genotype. METHODS: We performed a transverse, prospective and comparative study in patients with HCV infection in hemodialysis, continuous ambulatory peritoneal dialysis and patients in peritoneal dialysis, but with history of hemodialysis. Viral load was quantified with RT-PCR by using a commercial kit known as Amplicor HCV 2.0. Clinical variables studied were: age, gender, end stage renal disease etiology, modality and time in dialysis, transfusions, serum albumin, aminotransferases, blood urea nitrogen, and serum creatinine. RESULTS: Twenty four patients in dialysis with HCV infection entered into the study. Of these patients, 25% were on peritoneal dialysis, 29% on peritoneal dialysis with history of hemodialysis, and 46% were in hemodialysis. The average viral load (copies x 10(6)/mL) was 1.41 +/- 3.01. Viral load was lower in patients on peritoneal dialysis than in patients treated, or with history of hemodialysis (0.20 +/- 0.12 vs 2.04 +/- 0.88; p < 0.05). We observed no differences in viral load among patients with different viral genotypes. DISCUSSION: The average viral load of our patients in dialysis is lower than the levels usually observed in hepatitis C infected patients without end stage renal disease. The lower viral load in patients treated with peritoneal dialysis, and no history of hemodialysis, probably denotes lower risk of chronic liver disease in these subpopulation.

Functional properties of a truncated recombinant GIRK5 potassium channel.

Xenopus laevis oocytes codify a G-protein-activated inward rectifier potassium channel (GIRK5 or Kir3.5). Coinjection of other GIRKs, the muscarinic m2 receptor, or Gbetagamma protein cRNAs is required to observe functional GIRKx-GIRK5 heteromultimers in oocytes. Studies with GIRK2 isoforms have shown that the size of the amino or carboxyl terminus plays a crucial role on giving functional K(+) channels. In this work we studied the properties of a GIRK5 with 25 amino acids deleted toward its amino-terminal domain. Injection of GIRK5-Delta25 cRNA alone displayed large basal and transient inward rectifying currents in oocytes. The instantaneous currents reached a stationary level after a long duration voltage pulse (10 s). For this relaxation, fast (tau(1)) and slow (tau(2)) time constants were estimated at different voltages. Recovery from inactivation followed a monoexponential function (tau=0.95+/-0.07 s). By contrast with other inward rectifier channels, blockade of GIRK5-Delta25 by extracellular Ba(2+) was voltage-independent (K(d)=102+/-2 microM), suggesting the presence of a Ba(2+) site at the external channel vestibule. To confirm this hypothesis, the Ba(2+) sensitivity of two charged mutants GIRK5-Delta25(N129E) and GIRK5-Delta25(K157E) at each of the external loops was determined. GIRK5-Delta25(N129E) and GIRK5-Delta25(K157E) showed a 100-fold and 2-fold higher affinity to Ba(2+), respectively, supporting the existence of this Ba(2+) binding site.

Effect of aminoguanidine, a nitric oxide synthase inhibitor, on ocular infection with herpes simplex virus in Balb/c mice.

PURPOSE: To study the effect of aminoguanidine (AMG), an inhibitor of nitric oxide production, on the ocular infection of Balb/c mice with herpes simplex virus (HSV) type 1 strain F and HSV-2 strain G. METHODS: Animals were treated with different amounts of AMG (0.5, 0.1, and 0.05 mg/mouse) by topical application in the eye from postinfection (PI) days -2 through +5, considering 0 the day of infection. At different PI days, development of herpetic keratitis was evaluated in treated and control mice. RESULTS: Treated animals showed a dose-dependent increase in ocular disease after viral infection, compared with control animals. Viral titers in ocular washings were higher in AMG-treated mice (PI day 2, HSV-1: AMG 0.5 mg, 1.3 x 10(3) plaque-forming units (PFU)/ml; control, 0. 22 x 10(2) PFU/ml, P < 0.025). At PI day 3, control corneas had only scattered inflammatory cells, whereas those from treated animals showed a conspicuous infiltrate consisting primarily of neutrophils. Viral titers were also higher in brains of treated mice. These animals died earlier and in a greater proportion than control animals (percentage of mortality, PI day 12, HSV-1: AMG 0.5 mg, 40% +/- 4%; control, 18% +/- 3%, P < 0.05). CONCLUSIONS: These data indicate an inhibitory effect of nitric oxide on HSV ocular infection.