European study on orthopaedic status of haemophilia patients with inhibitors.

Development of inhibitors against factor VIII (FVIII) or factor IX (FIX) in haemophilia patients is one of the most serious complications of repeated exposure to replacement therapy and has major clinical and economic consequences. To evaluate the relationship between inhibitor status of haemophilia patients and their quality of life (QoL) and degree of arthropathy and to compare the orthopaedic status of patients with/without inhibitors. An observational, cross-sectional, case control study enrolling: group A (n = 38), males aged 14-35 years, with severe congenital haemophilia A or B who had inhibitors against FVIII/FIX >5 years; group B (n = 41), as group A, but aged 36-65 years and group C (n = 49), as group A, but without inhibitors. Socio-demographics: medical history, clinical characteristics and QoL were assessed. In groups A and B, 16% and 27% were hospitalized for orthopaedic procedures vs. 4% in group C. Patient mobility was also severely reduced in groups A and B, with 24% and 22% using wheelchairs vs. 4% in group C, and 50% and 51% needing a walking aid vs. 29% in group C. Significantly more joint pain was reported by patients in group A vs. those in group C; clinical/radiological orthopaedic scores were also worse in group A vs. group C. Significantly more joint abnormality was reported by patients in group A vs. group C. The burden of orthopaedic complications and the impact on QoL are more severe in haemophilia patients who have developed inhibitors than in those without inhibitors.

Effects of epidural anaesthesia-analgesia on intravenous anaesthesia with propofol.

The main purpose of this study was to demonstrate that the use of epidural anaesthesia-analgesia reduces the amount of propofol necessary to maintain surgical anaesthesia in dogs during ovariohysterectomy. The study was carried out on 28 bitches undergoing ovariohysterectomy with general anaesthesia using an intravenous infusion of propofol. Dogs were allocated to one of two groups. Group 1 received systemic buprenorphine together with epidural analgesia using lidocaine and buprenophine, and in Group 2 systemic buprenorphine only was given. The mean propofol infusion rate was significantly lower in dogs with epidural analgesia (P < 0.0001). In addition, the mean endotracheal extubation time was significantly longer in dogs with epidural analgesia (P < 0.0001). No significant differences were detected in any of the physiological parameters measured.

Inhibition of beta-amyloid toxicity by short peptides containing N-methyl amino acids.

Single N-methyl amino acid-containing peptides related to the central hydrophobic region beta16-20 (Lys-Leu-Val-Phe-Phe) of the beta-amyloid protein are able to reduce the cytotoxicity of natural beta1-42 in PC12 cell cultures. N-methyl phenylalanine analogs yield statistically significant increments in cell viability (Student's t-test < 0.01%) and are nontoxic in the same assay. These promising results indicate that these peptide molecules could be a starting point for the development of potential therapeutic compounds for the treatment of Alzheimer's disease.

Transplacental transfer of propofol in pregnant ewes.

Propofol is an injectable anaesthetic that is currently used both in veterinary and human medicine for the induction and maintenance of anaesthesia. Although little is known about the pharmacokinetics of propofol in fetuses, it is widely used in obstetric procedures, particularly in caesarean section. This study determines the pharmacokinetics of propofol in pregnant ewes in the last third of pregnancy, and placental transfer and pharmacokinetics in fetuses after the administration of a 6 mg/kg intravenous (i.v.) bolus (phase 1) or a 6 mg/kg i.v. bolus followed by continued infusion of 0.4 mg/kg/min. In ewes, the area under the blood concentration-time curve (AUC) and C(max) (8.6 mgh/mL and 9.5mg/mL, respectively) was higher than those of the fetus (1.6 mgh/mL and 1.19 mg/mL, respectively). The mean half-life was 0.5h in the dam and 1.1h in the fetus.

Prophylaxis in developed and in emerging countries.

The significant progress made in recent years in the safety and efficacy of both plasma-derived and recombinant factor concentrates has allowed treatment programmes to be developed that go beyond the simply curative or the treatment of the consequences of the disease (episodic or on-demand treatment), enabling the criteria of preventative medicine to be applied to congenital bleeding disorders. The aim of these programmes is to achieve constant, minimum levels of factor in patients above 1 or 2%, thereby converting severe haemophilia into moderate haemophilia.

Results of an orthopaedic survey in young patients with severe haemophilia in Spain.

This paper outlines the results obtained in a cross-sectional study of a group of young patients with severe haemophilia A and B. The primary aim of the study was to ascertain the level of orthopaedic complications in the group, the effects that these complications have on quality of life, and the medical resources used on these patients. The secondary aim was to relate their current orthopaedic state to the type of treatment received before the study. The study was carried out in 11 hospitals in Spain, where 70 severe haemophilia patients (factor VIII [FVIII] < 2%), with an average age of 21.6 and a median age of 22, and no inhibitors, were monitored. The percentage of patients suffering from articular complaints was 84.3% and 85.7% according to the Gilbert and Petterson scales, respectively. Furthermore, pain was reported in 16.1% of joints, most frequently in ankles. Using the SF-36 Health Survey, patients were observed to have a poorer quality of life relative to healthy controls. Despite high levels of both the coagulant factor given to patients and the financial resources dedicated to their treatment, the type of treatment (on demand and/or as secondary prophylaxis) provided to the young adults was found to be incapable of preventing haemophilic arthropathy and subsequent negative quality of life consequences.

Pilot testing of the 'Haemo-QoL' quality of life questionnaire for haemophiliac children in six European countries.

In a multinational working group, an instrument (Haemo-QoL) to assess quality of life in children/adolescents with haemophilia and their parents has been developed. In co-operation with haemophilia treatment centres in six European countries, approximately 10 children/adolescents with haemophilia per country and their parents were asked to participate in the pilot-testing. Both self-reported and parent-reported questionnaires were provided for two age-groups of children (4-16 years). Medical data was collected from physicians from patient files. Answers to open questions from participants (58 children and 57 parents) confirmed the content of 116 of the preliminary items. Cognitive debriefing revealed that the majority of the Haemo-QoL was rated favourably, but 29 questions were recommended to be omitted and several items to be reformulated. Preliminary psychometric testing of the revised 77 item questionnaire in the same sample showed acceptable reliability and validity, which will be examined in a subsequent study with a larger patient sample.

[Laparoscopic splenectomy in pediatric hematologic diseases]. / Esplenectomía laparoscópica en las enfermedades hematológicas pediátricas.

INTRODUCTION: Surgical acceptance of the laparoscopic splenectomy in the pediatric hematological disorders has improved due to safety, to relif postoperative pain, and to reduce the hospital stay. The aim of this study is present our surgical laparoscopic technique performed. MATERIAL AND METHODS: Eight patients, 6 spherocytosis (age ranged: 6-14 years, average age: 9.5 years; 5 girls and 1 boy) and 2 cases of autoimmune hemolytic anemia (both boys of 18 months and 4 years) underwent laparoscopic splenectomy. The Trias-Targarona technique has been performed placing the patient at the right lateral-decubitus position. Four trocars are placed, two of 5 mm in the posterior axillary line and epigastrium and other 2 trocars of 12 mm, in the middle of them. RESULTS: In all these 8 patients a laparoscopic splenectomy was completely performed. We had 2 cases with problems with the sac introduction. Cosmetic results have been excellent and we changed the 7-day antibiotic protocol for 2-day protocol. The surgical time has changed from 3 to 2 hours. CONCLUSIONS: With laparoscopic splenectomy we can give an adequate response to hematological disorders in children with a acceptable surgical time, an excellent cosmetic result with a minimum postoperative time and a very short hospital stay.

Role of calmodulin in the differentiation/activation of microglial cells.

In the present work the role of calmodulin (CaM) in regulating lipopolysaccharide (LPS)-induced microglial activation and in the spontaneous microglial differentiation has been investigated. We used pure rat microglial cell cultures to examine the effects of W13, a specific inhibitor of CaM, on microglial activation produced by LPS and the effect of CaM inhibition on microglial proliferation induced by the macrophage colony-stimulating factor (M-CSF). Microglial morphological transformation, inducible nitric oxide synthase (iNOS) activity and proliferating cell nuclear antigen (PCNA) immunostaining were determinate. Results show that CaM does not participate in the microglial increase of iNOS produced by LPS. In contrast, it is involved in spontaneous microglial ramification and in the activation of proliferation from quiescence. Multiple second-messenger pathways are involved in the transduction of signals initiated by LPS. The study of these mechanisms may allow us to extend our knowledge of the signals controlling the expression of these mediators.

The Ca2+/calmodulin system in neuronal hyperexcitability.

Calmodulin (CaM) is a major Ca2+-binding protein in the brain, where it plays an important role in the neuronal response to changes in the intracellular Ca2+ concentration. Calmodulin modulates numerous Ca2+-dependent enzymes and participates in relevant cellular functions. Among the different CaM-binding proteins, the Ca2+/CaM dependent protein kinase II and the phosphatase calcineurin are especially important in the brain because of their abundance and their participation in numerous neuronal functions. Therefore, the role of the Ca2+/CaM signalling system in different neurotoxicological or neuropathological conditions associated to alterations in the intracellular Ca2+ concentration is a subject of interest. We here report different evidences showing the involvement of CaM and the CaM-binding proteins above mentioned in situations of neuronal hyperexcitability induced by convulsant agents. Signal transduction pathways mediated by specific CaM binding proteins warrant future study as potential targets in the development of new drugs to inhibit convulsant responses or to prevent or attenuate the alterations in neuronal function associated to the deleterious increases in the intracellular Ca2+ levels described in different pathological situations.

BS-SEM evaluation of the tissular interactions between cortical bone and calcium-phosphate covered titanium implants.

The improvement of the reliability of the contact between the osseous tissues and the implant materials has been tested by recovering the metallic implants with ceramic materials, usually calcium phosphates. In our study, the calcium phosphate recovering layers were deposited by means of a pulsed-laser deposition technique. Our aim was to to evaluate the tissue interactions established between cortical bone and titanium implants covered by five different layers, ranging from amorphous calcium phosphate to crystalline hydroxyapatite, obtained by altering the parameters of the laser ablation process. The surgical protocol of the study consisted in the simultaneous implantation of the five types of implants in both the tibial dyaphisis of three Beagle dogs, sacrificed respectively one, two and three months after the last surgical procedures. After the sacrifice, the samples were submitted to a scheduled procedure of embedding in plastic polymers without prior decalcification, in order to perform the ultrastructural studies: scanning microscopy with secondary and backscattered electrons (BS-SEM). Our observations show that both in terms of the calcified tissues appearing as a response to the presence of the different coatings and of time of recovering, the implants coated with crystalline calcium phosphate layers by laser ablation present a better result than the amorphous-calcium-phosphate-coated implants. Moreover, the constant presence of chondroid tissue, related with the mechanical induction by forces applied on the recovering area, strongly suggests that the mechanisms implied in osteointegration are related to endomembranous, rather than endochondral ossification processes.

Treatment of children with haemophilia in Europe: a survey of 20 centres in 16 countries.

A survey was made of the current status of treatment of haemophilic boys at 20 centres in 16 European countries and includes approximately 1500 of the estimated 6500 haemophiliacs in the participating countries. Many mild haemophiliacs are not seen, or seen infrequently, at haemophilia centres and this requires study. Nine of 18 centres provide continuous prophylaxis to 80-100% of their patients, five centres provide it to 55-80% and the remaining four centres to 15-40% of the boys. The median dose given was 6240 U kg-1 year-1 (range 3120-7800). Four centres administered only recombinant concentrates to children with severe haemophilia A, while seven centres administered recombinant concentrates to 75-90% and the remaining centres to less than 50% of the boys (two centres < 10%). When asked for the choice of concentrate for a newly diagnosed boy with severe haemophilia A, all but one centre preferred recombinant concentrate. Most boys below 6 years received concentrates via a peripheral vein but three centres preferred a central venous line for 80-100% of the boys. Thirteen of 18 centres applied home treatment to 84-100% of the boys and the remaining five centres to 57-77% of the boys.

Estudio epidemiológico de la hemofilia en España / Epidemiological study of hemophilia in Spain

El objetivo de este estudio epidemiológico fue la caracterización de un grupo representativo de pacientes del territorio español afectos de hemofilia con relación a su situación diagnóstica, clínica, terapéutica y a las complicaciones de la enfermedad y de su tratamiento. Se llevó a cabo por medio de un cuestionario que se envió a todos los centros de tratamiento. Resultados: fueron remitidos 825 cuestionarios evaluables, lo que corresponde aproximadamente a un tercio de la población hemofílica española. El 88 por ciento de los hemofílicos padecían hemofilia A y el 12 por ciento hemofilia B. El 36 por ciento graves (nivel inferior al 2 por ciento). La gran mayoría de pacientes estaban siguiendo un programa de tratamiento a demanda (79 por ciento). Un reducido número seguía, en el momento del registro, un tratamiento de profilaxis secundaria (17 por ciento), y tan solo un 3,4 por ciento seguía un programa de profilaxis primaria. El consumo medio de factores por paciente durante el año 1995, fue de 45.945 UI, lo que correspondería a una media de 706 Ul/kg/año. El 30 por ciento de los pacientes presentaban una o más articulaciones con algún grado de afectación. Los pacientes graves presentaban un total de 627 articulaciones afectadas de media por paciente. El 7,3 por ciento del total presentaba o habían presentado inhibidor. El 27 por ciento tenían anticuerpos anti-VIH. Los anticuerpos de la hepatitis C estaban presentes en el 61 por ciento. El antígeno HBs fue positivo en el 4 por ciento. (AU)

The orthopaedic status of severe haemophiliacs in Spain.

This paper provides an outline of the results obtained in a cross-sectional study conducted primarily with the aim of ascertaining orthopaedic complications in a group of young severe A and B haemophiliacs, the effects which these complications have on the medical resources used with these patients, and the impact of severe haemophilia on their quality of life. Its secondary aim was to link their current orthopaedic status to the type of treatment they had received prior to the study. Eleven Spanish hospitals took part in this study, monitoring 70 severe haemophiliacs (FVIII:C <2%) without inhibitors who had a mean age of 21.6 years and a median age of 22. Retrospective data collected from birth to the conclusion of the study were used and, for certain variables, data from the last 12 months. The type of treatment given had been on-demand treatment, together with prophylaxis of variable time periods, which in 32 cases (45.7%) were prolonged (>6 months). In 40 cases (57.1%) the patients underwent one or more periods of prophylaxis. Thirty-three patients (47.8%) had over 1000 days of administration of factors VIII and IX. The analysis of the total study group reveal an average of 348 bleeding episodes per patient. The findings of this study revealed that 84.3% of these patients suffer from articular complaints on the Gilbert scale, and 85.7% on the Pettersson scale. In addition, pain was reported in 16.1% of the joints, the most frequently affected being the ankle joints. Twenty-six patients (37%) had undergone orthopaedic surgery from the time of birth to the conclusion of the study. The quality of life of the severe haemophiliacs reviewed seems to have been affected. During the last 12 months, there were 216 outpatient haematological visits and 176 orthopaedic-rehabilitation visits, as well as 12 radiological explorations and two hospitalizations. During these 12 months, medical expenditure totalled $55 473 per patient per year, the most important item being factor concentrates VIII/IX ($54 119 per patient per year). The type of treatment given to these patients (on-demand and/or as secondary prophylaxis) was found to be incapable of preventing haemophilic arthropathy or guaranteeing an acceptable quality of life, although both the administration of coagulant factor concentrate to such patients and the financial resources dedicated to their treatment was very high. Consequently, a strategic change is to be considered for the prevention of haemophilic arthropathy, based on the introduction of replacement treatment involving continuous administration of factors VIII and IX in primary prophylaxis regimens from the first years of life.

Hepatitis G virus infection markers (RNA and anti-E2 antibodies) in a multicenter cohort of hemophiliacs.

BACKGROUND AND OBJECTIVE: To investigate the prevalence and evolution of hepatitis G virus (HGV) infection in hemophiliacs and to correlate evolution of HGV infection markers with immunologic parameters in those patients co-infected with HIV. DESIGN AND METHODS: HGV RNA and anti-E2 antibodies were studied in 124 patients. Serial samples were drawn every 4 months from 1992 to 1996. Lymphocyte subsets including T-helper lymphocytes, T-suppressor lymphocytes, T-cytotoxic lymphocytes, activated T-lymphocytes and natural killer cells were analyzed. RESULTS: Prevalences were 22.6% for HGV RNA and 18.5% for anti-E2. Four patients had both HGV RNA and anti E2, so the overall prevalence of HGV infection in hemophiliacs was 37.9% (11.5% in 200 controls, p<0.0001). After a median follow-up of 36.6 months 20 patients remained HGV RNA positive, whereas HGV RNA had cleared in 8, with an actuarial probability of clearance at 36 months of 34.6%. Only 2 patients developed anti-E2 antibodies. Four patients cleared anti-E2, with an actuarial probability at 36 months of 24.8%. In patients with HIV infection, both lower CD4+ lymphocyte count (p=0.01) or higher CD8+ lymphocyte count (p=0.03) showed predictive value for probability of clearing HGV-RNA. CD4+/CD8+ ratio (p=0.002) was the only variable included in the best model for HGV-RNA disappearance. INTERPRETATION AND CONCLUSIONS: A more accurate estimation of the prevalence of HGV infection can be achieved with the determination of both HGV RNA and anti-E2. Anti-E2 response can be undetectable or transitory after disappearance of HGV-RNA, giving therefore rise to the possibility of underestimating HGV prevalence with currently diagnostic methods. In HIV-positive patients, cellular immune function seems to be involved in the resolution of HGV infection, following the significant correlation found between clearance of HGV-RNA and CD4+/CD8+ lymphocyte populations.

Comparative study of the distribution of calmodulin kinase II and calcineurin in the mouse brain.

The Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and the phosphatase calcineurin (CaN) are Ca(2+)/calmodulin-binding proteins that are very abundant in the central nervous system. In the mammalian brain, CaMKII is composed by the association of several similar subunits at different ratios produced by four different genes. CaN is composed of two different subunits produced by two genes. We selected the most abundant subunits of each enzyme in the rodent brain, CaMKII alpha and CaN A, and compared their pattern of expression in the mouse brain by using in situ hybridization histochemistry and immunohistochemistry. We found that CaMKII and CaN were mainly expressed in cerebral cortex, hippocampus, and striatum and that low levels of expression were observed in midbrain and brainstem. CaN was also expressed in cerebellum. In the cell, the kinase and the phosphatase were detected in the perikarya, the neuronal processes, and the nucleus. The present study shows that all the regions of the mouse brain in which CaMKII is expressed also show CaN expression. This fact is consistent with the presence of common substrates for both enzymes or with a regulatory action of one versus the other. The lack of correspondence in the cerebellum could be explained by the fact that the major subunit of the kinase in this brain region is CaMKII beta.

The Ca2+/calmodulin signaling system in the neural response to excitability. Involvement of neuronal and glial cells.

Ca2+ plays a critical role in the normal function of the central nervous system. However, it can also be involved in the development of different neuropathological and neurotoxicological processes. The processing of a Ca2+ signal requires its union with specific intracellular proteins. Calmodulin is a major Ca(2+)-binding protein in the brain, where it modulates numerous Ca(2+)-dependent enzymes and participates in relevant cellular functions. Among the different calmodulin-binding proteins, the Ca2+/calmodulin-dependent protein kinase II and the phosphatase calcineurin are especially important in the brain because of their abundance and their participation in numerous neuronal functions. We present an overview on different works aimed at the study of the Ca2+/calmodulin signalling system in the neural response to convulsant agents. Ca2+ and calmodulin antagonists inhibit the seizures induced by different convulsant agents, showing that the Ca2+/calmodulin signalling system plays a role in the development of the seizures induced by these agents. Processes occurring in association with seizures, such as activation of c-fos, are not always sensitive to calmodulin, but depend on the convulsant agent considered. We characterized the pattern of expression of the three calmodulin genes in the brain of control mice and detected alterations in specific areas after inducing seizures. The results obtained are in favour of a differential regulation of these genes. We also observed alterations in the expression of the Ca2+/calmodulin-dependent protein kinase II and calcineurin after inducing seizures. In addition, we found that reactive microglial cells increase the expression of calmodulin and Ca2+/calmodulin-dependent protein kinase II in the brain after seizures.

[Lupus anticoagulant: complete automation (ACL-Futura) and diagnostic algorithm]. / Anticoagulante lúpico: automatización completa (ACL-Futura) y algoritmo diagnóstico.

PURPOSE: The technical and clinical evaluation of a new laboratory profile with six tests, for the realization of the assay called "lupic anticoagulant" with the help of ACL-Futura analyzer (Intrumentation Laboratory). RESULTS: The within-between day imprecision of the tests that compose the profile: APTT-Diluted, APTT-D Mix, LacScreen, LacScreen Mix, LacConfirm and LacConfirm Mix, are between 2.87%-11.61% with controls, this imprecision is lowest with patients. A study of bilirrubin and lipemia interferences is presented. The practicability study present the technical, time consuming difficulty, and shows that the cost of the screening test is about 1136 ptas (6.83 euros) and with the confirmative test is 2766 ptas (16.62 euros). The clinical study describes our preliminary results with the application of this new profile for almost 2 years and six clinical cases are presented. CONCLUSIONS: The good technical and clinical results of the evaluation of the new profile proposed to detect the positive lupic anticoagulant, in addition to the fully automated assay with the ACL-Futura analyzer, validate the whole method to resolve the increased demand of these parameters.

Experiences with continuous infusion of recombinant activated factor VII.

A questionnaire was sent to 28 haemophilia treatment centres known to have used recombinant activated factor VII (rFVIIa; NovoSeven, Novo Nordisk, Bagsvaerd, Denmark), to collect data on continuous infusion of this product. This mode of administration was recently introduced for rFVIIa but there are several questions which remain to be answered in order to optimize this technique. Of the 26 responding centres, 14 had used rFVIIa in continuous infusion for 40 treatment episodes over a total of 283 days. In most of the cases the treatment was targeted at a factor VII level of 10 IU/ml, monitored by the one-stage clotting assay. This seemed to be adequate for most of the haemorrhagic and surgical procedures. Pretreatment pharmacokinetic evaluation was performed in only a minority of the cases but is probably of great importance given the wide variation observed in the clearance values. A strategy was necessary to prevent local thrombophlebitis, at least for infusions in peripheral veins; parallel infusion of heparin, saline or dextrose-saline proved effective. The question of optimal monitoring needs further attention. Haemorrhagic complications were significantly less frequent when treatment was combined with the antifibrinolytic tranexamic acid.