Investigation of the relationship between thrombophilic disorders and brain white matter lesions in migraine with aura.

BACKGROUND: Migraine is associated with several genetic or acquired comorbidities. Studies conducted in recent years emphasize that the frequency of thrombophilia is high in migraine, especially migraine with aura (MA). Similarly, the presence of white matter lesions (WMLs) on brain magnetic resonance imaging (MRI) scans has been associated with migraine for many years. OBJECTIVE: Based on the knowledge that both WMLs and thrombophilia variants are frequently observed in MA, we aimed to investigate whether there is a relationship between genetic thrombophilia and the presence of WMLs in these patients. METHODS: The levels of proteins S and C, antithrombin III activities, activated protein C (APC) resistance, antiphospholipid immunoglobulin G/immunoglobulin M (IgG/IgM) and anticardiolipin IgG/IgM antibodies were investigated in 66 MA patients between the ages of 18 and 49 years who presented no cardiovascular risk factors. The presence of WMLs and the Fazekas grade was determined from the brain magnetic resonance imaging (MRI) scans' T2-weighted and fluid-attenuated inversion recovery (FLAIR) sequence taken from the patients. The rates of WMLs were compared in patients with and without thrombophilia. RESULTS: Thrombophilia was detected in 34.8% of the patients, and 27.3% were determined to have WMLs in brain MRI scans. The WMLs were detected in 23.3% of the patients without thrombophilia, in 34.8% of those with thrombophilia, and in 50% of the subjects with multiple thrombophilia disorders. Among the thrombophilia disorders, only APC resistance was significantly more common in patients with WMLs. CONCLUSION: The results of the present study showed that thrombophilia may be a mechanism that should be investigated in the etiology of increased WMLs in MA.

ANTECEDENTES: La migraña se asocia con una serie de comorbilidades genéticas o adquiridas. Los estudios realizados en los últimos años destacan que la frecuencia de trombofilia es elevada en la migraña, especialmente en la migraña con aura (MA). De manera similar, la presencia de lesiones de la sustancia blanca (LSB) en las imágenes por resonancia magnética (RM) del cerebro se ha asociado con la migraña hace muchos años. OBJETIVO: Con base en la información de que se suelen observar tanto LSB como variantes de la trombofilia en MA, nuestro objetivo fue investigar si existe una relación entre la trombofilia genética y la presencia de LSB en estos pacientes. MéTODOS: Se investigaron los niveles de proteína S y de proteína C, actividades de antitrombina III, resistencia a la proteína C activada (PCA), anticuerpos antifosfolípidos inmunoglobulina G/inmunoglobulina M (IgG/IgM) y anticuerpos anticardiolipina IgG/IgM en 66 pacientes con MA entre 18 y 49 años que no presentaban factores de riesgo cardiovascular. Se determinaron la presencia de LSB y el grado de Fazekas a partir de imágenes por RM del cerebro en la secuencia ponderada en T2 y recuperación de la inversión atenuada de fluido (fluid-attenuated inversion recovery, FLAIR, en inglés) obtenidas de los pacientes. Se compararon las tasas de LSB en pacientes con y sin trombofilia. RESULTADOS: Se detectó trombofilia en el 34,8% de los pacientes y LSB en el 27,3%. Las LSB estuvieron presentes en el 23,3% de los pacientes sin trombofilia, en el 34,8% de los que tenían trombofilia, y en el 50% de los que tenían múltiples trastornos trombofílicos. La resistencia a la PCA fue significativamente más común en aquellos pacientes con LSB. CONCLUSIóN: Los resultados del presente estudio mostraron que la trombofilia puede ser un mecanismo que debe investigarse en la etiología del aumento de LSB en MA.

Duodenal mucosa of untreated celiac disease patients has altered expression of the GAS6 and PROS1 and the negative regulator tyrosine kinase TAM receptors subfamily.

Celiac disease (CD) is an immune-driven disease characterized by tissue damage in the small intestine of genetically-susceptible individuals. We evaluated here a crucial immune regulatory pathway involving TYRO3, AXL, and MERTK (TAM) receptors and their ligands PROS1 and GAS6 in duodenal biopsies of controls and CD patients. We found increased GAS6 expression associated with downregulation of PROS1 and variable TAM receptors levels in duodenum tissue of CD patients. Interestingly, CD3+ lymphocytes, CD68+, CD11c+ myeloid and epithelial cells, showed differential expressions of TAM components comparing CD vs controls. Principal component analysis revealed a clear segregation of two groups of CD patients based on TAM components and IFN signaling. In vitro validation demonstrated that monocytes, T lymphocytes and epithelial cells upregulated TAM components in response to IFN stimulation. Our findings highlight a dysregulated TAM axis in CD related to IFN signaling and contribute to a deeper understanding of the pathophysiology of CD.

Antiviral potential of diminazene aceturate against SARS-CoV-2 proteases using computational and in vitro approaches.

Diminazene aceturate (DIZE), an antiparasitic, is an ACE2 activator, and studies show that activators of this enzyme may be beneficial for COVID-19, disease caused by SARS-CoV-2. Thus, the objective was to evaluate the in silico and in vitro affinity of diminazene aceturate against molecular targets of SARS-CoV-2. 3D structures from DIZE and the proteases from SARS-CoV-2, obtained through the Protein Data Bank and Drug Database (Drubank), and processed in computer programs like AutodockTools, LigPlot, Pymol for molecular docking and visualization and GROMACS was used to perform molecular dynamics. The results demonstrate that DIZE could interact with all tested targets, and the best binding energies were obtained from the interaction of Protein S (closed conformation -7.87 kcal/mol) and Mpro (-6.23 kcal/mol), indicating that it can act both by preventing entry and viral replication. The results of molecular dynamics demonstrate that DIZE was able to promote a change in stability at the cleavage sites between S1 and S2, which could prevent binding to ACE2 and fusion with the membrane. In addition, in vitro tests confirm the in silico results showing that DIZE could inhibit the binding between the spike receptor-binding domain protein and ACE2, which could promote a reduction in the virus infection. However, tests in other experimental models with in vivo approaches are needed.

Association of Pre-S/S and Polymerase Mutations with Acute and Chronic Hepatitis B Virus Infections in Patients from Rio de Janeiro, Brazil.

Several hepatitis B virus (HBV)-related factors, including the viral load, genotype, and genomic mutations, have been linked to the development of liver diseases. Therefore, in this study we aimed to investigate the influence of HBV genetic variability during acute and chronic infection phases. A real-time nested PCR was used to detect HBV DNA in all samples (acute, n = 22; chronic, n = 49). All samples were sequenced for phylogenetic and mutation analyses. Genotype A, sub-genotype A1, was the most common genotype in the study population. A total of 190 mutations were found in the pre-S/S gene area and the acute profile revealed a greater number of nucleotide mutations (p < 0.05). However, both profiles contained nucleotide mutations linked to immune escape and an increased risk of hepatocellular carcinomas (acute, A7T; chronic, A7Q). Furthermore, 17 amino acid substitutions were identified in the viral polymerase region, including the drug resistance mutations lamivudine and entecavir (rtL180M), with statistically significant differences between the mutant and wild type strains. Owing to the natural occurrence of these mutations, it is important to screen for resistance mutations before beginning therapy.

Coagulation profile of COVID-19 patients admitted to the ICU: An exploratory study.

BACKGROUND: Coagulation abnormalities in COVID-19 patients have not been addressed in depth. OBJECTIVE: To perform a longitudinal evaluation of coagulation profile of patients admitted to the ICU with COVID-19. METHODS: Conventional coagulation tests, rotational thromboelastometry (ROTEM), platelet function, fibrinolysis, antithrombin, protein C and S were measured at days 0, 1, 3, 7 and 14. Based on median total maximum SOFA score, patients were divided in two groups: SOFA ≤ 10 and SOFA > 10. RESULTS: Thirty patients were studied. Some conventional coagulation tests, as aPTT, PT and INR remained unchanged during the study period, while alterations on others coagulation laboratory tests were detected. Fibrinogen levels were increased in both groups. ROTEM maximum clot firmness increased in both groups from Day 0 to Day 14. Moreover, ROTEM-FIBTEM maximum clot firmness was high in both groups, with a slight decrease from day 0 to day 14 in group SOFA ≤ 10 and a slight increase during the same period in group SOFA > 10. Fibrinolysis was low and decreased over time in all groups, with the most pronounced decrease observed in INTEM maximum lysis in group SOFA > 10. Also, D-dimer plasma levels were higher than normal reference range in both groups and free protein S plasma levels were low in both groups at baseline and increased over time, Finally, patients in group SOFA > 10 had lower plasminogen levels and Protein C ​​than patients with SOFA <10, which may represent less fibrinolysis activity during a state of hypercoagulability. CONCLUSION: COVID-19 patients have a pronounced hypercoagulability state, characterized by impaired endogenous anticoagulation and decreased fibrinolysis. The magnitude of coagulation abnormalities seems to correlate with the severity of organ dysfunction. The hypercoagulability state of COVID-19 patients was not only detected by ROTEM but it much more complex, where changes were observed on the fibrinolytic and endogenous anticoagulation system.

Vacunas para SARS-CoV-2: diferentes estrategias de los desarrollos en curso / Vaccines for SARS-CoV-2: different strategies of ongoing developments

El objetivo de este artículo es proporcionar una guía que sirva para la interpretación y seguimiento de los esfuerzos que se están desarrollando en todo el mundo con el objetivo de obtener una vacuna que pueda generar inmunidad contra el nuevo coronavirus SARS-CoV-2 de 2019, el agente causante de la enfermedad por coronavirus denominada COVID-19. Cinco meses después de haber sido detectada la enfermedad, ya hay 102 vacunas en distintos estadios de desarrollo, registradas por la Organización Mundial de la Salud (OMS), correspondientes a 8 plataformas vacunales con diferentes estrategias, y todos los días aparecen nuevas. Esto representará un enorme desafío de organismos internacionales, para la evaluación, comparación y selección de aquellas que cumplan con los criterios regulatorios indispensables de seguridad y eficacia y que, por otro lado, puedan ser producidas en cantidades suficientes para abastecer la demanda mundial. (AU)

The objective of this article is to provide a guide to help the interpretation and monitoring the efforts that are being carried out worldwide to obtain a vaccine that will be able to generate immunity against the new 2019 SARS-CoV-2 coronavirus, the viral agent causes the disease named COVID-19. Five months after the disease was detected, there are already 102 vaccines at different stages of development, registered by World Health Organization (WHO), corresponding to 8 vaccination platforms base on different strategies, and every day new ones appear. This will represent a huge challenge for international organizations, to evaluate, compare and selects those that will meet the essential regulatory criteria of safety and efficacy and that, would be able to be produced in enough quantities to supply the worldwide demand. Key words: SARS-Cov-2 vaccine, vaccine platform, COVID-19 strategy, attenuated virus, viral vector, viral proteins, viral DNA, viral RNA, nucleic acids, viral like particles, WHO. (AU)

Spinal anesthesia for c-section in patients with protein S deficiency: case report and literature review / Anestesia espinal para cesárea en paciente con deficiencia de proteína S: informe de caso y revisión de la literatura

Abstract Introduction: Congenital protein S deficiency is a very rare disease in the population. In pregnant women it is associated with spontaneous abortion and fetal death, among other complications. Case presentation: We present the case of a 32-year-old multigravida with a 36-week pregnancy, with thromboprophylaxis with enoxaparin from the 4th week of gestation and with a diagnosis of thrombophilia-due to functional protein S deficiency-which was intervened with elective c-section under spinal anesthesia. In addition, a review of the relevant literature was conducted. Discussion: The risk of venous thromboembolism is approximately 4 to 5 times greater during gestation, and the recommendation of thromboprophylaxis in low-risk thrombophilia is based on the presence of associated risk factors. In patients receiving low molecular weight heparin (LMWH) as thromboprophylaxis, an interval of at least 12 hours after the last dose of LMWH before neuropsy and restarting the next dose after at least 4hours of spinal technique use is recommended. Conclusion: Neuroaxial techniques should be individualized and receive pre and postpartum thromboprophylaxis. In addition, non-pharmacological thromboprophylaxis measures in the perioperative period should be considered. Spinal anesthesia was effective and safe in this patient.

Resumen Introducción: La deficiencia congénita de proteína S es una enfermedad muy rara en la población. En gestantes está asociada a aborto espontáneo y muerte fetal, entre otras complicaciones. Presentación del caso: Presentamos el caso de una multigesta de 32 años con embarazo de 36 semanas, con tromboprofilaxis con enoxaparina desde la semana cuarta de gestación y con diagnóstico de trombofilia -por deficiencia de proteína S funcional-, la cual fue intervenida con cesárea electiva bajo anestesia espinal. Además, se realizó revisión de la literatura al respecto. Discusión: El riesgo de tromboembolismo venoso es aproximadamente 4 a 5 veces mayor durante la gestación, y la recomendación de tromboprofilaxis en trombofilias de bajo riesgo se basa en la presencia de factores de riesgo asociados. En pacientes que reciben Heparinas de Bajo Peso Molecular (HBPM) como tromboprofilaxis, se recomienda un intervalo de al menos 12 horas después de la última dosis de HBPM antes de la punción del neuroeje, y reiniciar la siguiente dosis después de al menos 4 horas de uso de la técnica espinal. Conclusión: Las técnicas neuroaxiales deben ser individualizadas y recibir tromboprofilaxis pre y posparto. Además, se deben tener en cuenta las medidas de tromboprofilaxis no farmacológicas en el periodo perioperatorio. La anestesia espinal fue efectiva y segura en esta paciente.

Nefrolitotomía percutánea en pacientes con trastornos de la coagulación / Percutaneous Nephrolithotomy in Patients with Bleeding Disorders

Objetivo Reportar un caso de nefrolitotomía percutánea en paciente con deficiencia de proteína C y S. Introducción Los pacientes con déficit de proteína C y S tienen un riesgo alto de eventos tromboembólicos reportándose tasas de hasta el 6% y 8,4% respectivamente. Reporte de Caso Paciente femenina de 43 años con antecedente de deficiencia de proteína C y S, anticoagulación crónica con warfarina por trombosis venosa profunda (TVP), clínicamente con cuadro de cólico reno ureteral derecho y hematuria, la tomografía de vías urinarias mostró un cálculo coraliforme completo derecho. Fue llevada a nefrolitotomía percutánea derecha previa terapia puente con enoxaparina, el acceso percutáneo fue a través del cáliz inferior. Debido a que no fue posible acceder a los cálculos del cáliz medio y superior y que en la institución donde se realizó el procedimiento no se cuenta con nefroscopio flexible, se decidió realizar una segunda punción en cáliz superior, dejando a la paciente libre de cálculos. Se reinició la anticoagulación plena a las 12 horas del postoperatorio sin presentar sangrado asociado. Discusión Los pacientes con déficit de proteína C y S tienen un riesgo alto para eventos tromboembólicos. Kefer y col., realizaron un estudio en el que evaluaron la eficacia de la terapia puente en pacientes llevados a NLP, encontrando que la anticoagulación con warfarina puede suspenderse 5 días antes y reiniciarse 5 días después del procedimiento quirúrgico sin necesidad de terapia puente con enoxaparina. En la actualidad, las recomendaciones dadas por la Sociedad Americana de Urología, indican realizar la terapia puente mediante un grupo multidisciplinario. Resultados El déficit de proteína C y S corresponde a una entidad, con una prevalencia muy baja y condiciona el requerimiento de anticoagulantes orales de forma indefinida. Fue posible realizar una intervención quirúrgica sin complicaciones hemorrágicas ni tromboembólicas con el uso de terapia puente prequirúrgica.

Objective To report a case of percutaneous nephrolithotomy in patients with protein C and S deficiency. Introduction Patients with protein C and S deficiency have a high risk of thromboembolic events reporting rates of 6% and 8.4%, respectively. Case Report A 43-year-old female patient with a history of protein C and S deficiency with chronic warfarin anticoagulation for deep venous thrombosis (DVT). CT scan with full right staghorn calculi. Enoxaparin was administered bridge therapy. She was taken to right percutaneous nephrolithotomy, access was through the lower calyx. Because it was not possible to access the calculus of the middle and upper calyx it was necessary to perform a second puncture in the upper calyx, leaving the patient free of calculus. Full anticoagulation was resumed at 12 hours postoperatively without associated bleeding. Discussion Patients with protein C and S deficits are at high risk for thromboembolic events. Kefer et al. conducted a study evaluating the efficacy of bridge therapy in patients on NLP, finding that warfarin anticoagulation can be discontinued 5 days earlier and restarted 5 days after the surgical procedure without the need for enoxaparin bridging therapy. Results The protein C and S deficiency corresponds to an entity, with a very low prevalence and conditions the requirement of oral anticoagulants indefinitely. It was possible to perform a surgical procedure without hemorrhagic or thromboembolic complications.

Déficit de proteína S en un caso pediátrico de trombosis de senos venosos cerebrales / Protein S deficiency in a pediatric case of cerebral venous sinus thrombosis

RESUMEN La trombosis venosa cerebral (TVC) representa el 0,5 % de todos los eventos vasculares cerebrales a nivel mundial; es una entidad compleja y de difícil diagnóstico, dada la variabilidad en el modo de presentación, el pronóstico y la diversidad de condiciones médicas que la originan. Ocurre por obstrucción del drenaje venoso cerebral. Su principal manifestación en lactantes y niños son las convulsiones. Este es el caso de una TVC por déficit de proteína S en un paciente pediátrico, el objetivo de este caso es destacar la importancia de la sospecha e identificación temprana de la enfermedad, así como la búsqueda del factor desencadenante. La TVC es una entidad grave, es potencialmente tratable y su pronóstico dependerá de la oportunidad del diagnóstico e intervención.

SUMMARY Cerebral venous sinus thrombosis represents 0.5 % of all cases worldwide. Is a complex pathology cause the variability in presentation mode, prognosis and different medical conditions that cause it. The cause is the obstruction of cerebral venous drainage, the main manifestation in infants and children are seizures. This is the case of cerebral venous sinus thrombosis in a pediatric patient with protein S deficiency, the goal of this case is to recognize the importance of suspicion and early identification of the disease and the search of the trigger. The cerebral venous sinus thrombosis is a serious entity, but potentially treatable and prognosis depend on the timing of diagnosis and intervention.

Subcellular localisation of FLAG tagged enzymes of the dynamic protein S-palmitoylation cycle of Trypanosoma cruzi epimastigotes.

Dynamic S-palmitoylation of proteins is the addition of palmitic acid by zDHHC palmitoyl transferases (PATs) and depalmitoylation by palmitoyl protein thioesterases (PPTs). A putative PAT (TcPAT1) has been previously identified in Trypanosoma cruzi, the etiological agent of Chagas disease. Here we analyse other 14 putative TcPATs and 2 PPTs in the parasite genome. T. cruzi cell lines expressing TcPATs and TcPPTs plus a FLAG tag at the C terminus were produced for most enzymes, with positive detection by indirect immunofluorescence. Overexpressed TcPATs were mostly found as single spots at the parasite anterior end, while the TcPPTs were dispersed throughout the parasite body.

Subcellular localisation of FLAG tagged enzymes of the dynamic protein S-palmitoylation cycle of Trypanosoma cruzi epimastigotes

Dynamic S-palmitoylation of proteins is the addition of palmitic acid by zDHHC palmitoyl transferases (PATs) and depalmitoylation by palmitoyl protein thioesterases (PPTs). A putative PAT (TcPAT1) has been previously identified in Trypanosoma cruzi, the etiological agent of Chagas disease. Here we analyse other 14 putative TcPATs and 2 PPTs in the parasite genome. T. cruzi cell lines expressing TcPATs and TcPPTs plus a FLAG tag at the C terminus were produced for most enzymes, with positive detection by indirect immunofluorescence. Overexpressed TcPATs were mostly found as single spots at the parasite anterior end, while the TcPPTs were dispersed throughout the parasite body.

Trombosis de senos venosos cerebrales, una manifestación inusual de déficit de proteína S en paciente joven / Cerebral venous sinus thrombosis, an unusual manifestation of protein s deficiency in young patients

Resumen La trombosis venosa cerebral (TSVC) es un tipo de accidente cerebrovascular (ACV) que involucra el lado venoso de la circulación cerebral, incluye trombosis de los senos venosos durales y/o de las venas corticales y profundas del cerebro, es una causa poco común 0,5-1 % de todos los accidentes cerebrovasculares, con una prevalencia estimada en el rango entre 0. 22 a 1,23 / 100.000 / año. Los factores de riesgo para TSVC, están generalmente divididos en riesgos adquiridos (por ejemplo: cirugía, trauma, embarazo, puerperio, síndrome antifosfolípido, cáncer, hormonas exógenas) y los riesgos genéticos (trombofilia hereditaria). Los factores de riesgo más ampliamente estudiados para TSVC incluyen estados protrombóticos, las trombofilias heredadas asociadas con TSVC incluyen deficiencias de antitrombina, proteína C, proteína S (PS), mutación del factor V Leiden y la mutación del gen 20210 de protrombina. La prevalencia del déficit de PS, oscila entre un 0,02 y un 0,03 % en la población general y aumenta hasta un 2 % en pacientes no seleccionados con trombosis. Con una mortalidad cercana al 9 %. El manejo es usualmente médico. Se cita el caso de una paciente de 28 años de edad, con cuadro clínico de cefalea de 1 mes de evolución, con hallazgos en neuroimagen de trombosis de senos transverso y sigmoideo izquierdo con déficit de proteína S.

Summary Cerebral venous thrombosis (TSVC) is a type of stroke (CVA) involving the venous side of the cerebral circulation, including thrombosis of the dural venous sinuses and / or cortical and deep veins of the brain, is a rare cause 0.5-1% of all strokes, with an estimated range between 0. 22 to 1.23 / 100,000 / year prevalence. Risk factors for TSVC, are generally divided into acquired risks (eg, surgery, trauma, pregnancy, postpartum, antiphospholipid syndrome, cancer, exogenous hormones) and genetic risks (hereditary thrombophilia). The most widely studied factors TSVC risk include prothrombotic states, inherited thrombophilia associated with deficiencies TSVC include antithrombin, protein C, protein S (PS), mutation of factor V Leiden mutation and prothrombin 20210 gene. The prevalence of PS deficit ranges between 0.02 and 0.03% in the general population and increases up to 2% in unselected patients with thrombosis. With close to 9% mortality. The operation is usually doctor. It cites the case of a 28-year-old, with clinical symptoms of headache 1 month of evolution with neuroimaging findings transverse sinus thrombosis and left sigmoid with protein S deficiency.

Accidente cerebrovascular en un adulto joven con deficiencia de proteína S y foramen oval patente: reporte de caso / troke in a young adult with protein S deficiency and patent foramen ovale: case report

Se reporta el caso de una mujer joven de 38 años de edad, con accidente cerebrovascular isquémico agudo y deficiencia de proteína S más foramen oval patente. La paciente acudió al servicio de emergencia por presentar hemiparesia derecha y disartria, de forma súbita. Al examen físico se evidenció marcada disminución de la fuerza muscular en hemicuerpo derecho con signo de Babinski positivo. La tomografía cerebral mostró una hipodensidad cortico-subcortical témporo-parietal izquierda y la angioresonancia confirmó signos de infarto. La ecocardiografía transtorácica evidenció foramen oval patente y en los estudios de hipercoagulabilidad se encontró deficiencia de la Proteína S.

We report the case of 38-year-old woman presenting with an ischemic cerebrovascular accident, protein S deficiency and patent foramen ovale. The patient attended the emergency room for presently a sudden onset of right hemiparesis and dysarthria. The physical examination revealed marked palsy on the right side with positive Babinski sign. The cerebral CT-scan revealed a cortico-sub cortical left temporal-parietal hypo density, and the angio magnetic resonance confirmed the presence of cerebral infarct. A trans thoracic echocardiography revealed the presence of a patent foramen ovale and laboratory tests showed deficiency of S protein.

Anticoagulant proteins in a population of Mexican mestizo donors.

BACKGROUND: To determine the activity of antithrombin (AT), protein C (PC), and protein S (PS), as well as the frequency of deficiencies of these proteins in a population of healthy Mexican mestizo blood donors. METHODS: AT, PC, and PS were determined from 1,502 plasma samples of healthy blood donors by using commercial kits in a coagulometer 4 STA (Diagnostica Stago, Asnières, France). RESULTS: A total of 741 women and 761 men were under study. They were divided into age range groups (18-24, 25-34, 35-44, 45-54, and 55-64 years). Activity of AT, PC, and PS was determined. For AT, activity values were specific for each age group according to gender when it had to do with PS, as well as when PC was determined. Frequencies of AT, PC, PS, and activated PC resistance activity deficiencies were obtained from reference levels (RLs) and average levels of this study. Differences were found between both frequencies for AT, PC, and PS, and the average levels obtained were used in this study. The frequencies of the activity deficiencies obtained through the values gotten in this population were: AT, 0.6%; PC, 1.06% (which is higher than the one obtained using the RLs described by commercial kits 0.33% and 0.66%, respectively); and PS, 1% (which is less than 4.5%). CONCLUSIONS: It is necessary to know the characteristics and biological behavior of the coagulation proteins in the Mexican population because the RLs used have been established for populations that are genetically different.

Construction and functional identification of a hepatitis B virus S protein small hairpin RNA recombinant adenovirus.

Hepatitis B virus S protein (HBs) plays an important role in hepatocellular carcinoma progression. However, to date, no direct and effective methods exist to research the function of HBs. Here, we combined the technology of RNA interference with recombinant adenovirus, constructed a recombinant adenovirus-expressing small hairpin RNA of HBs, and infected HepG2.2.15 cells. Then, reverse transcription-polymerase chain reaction (RT-PCR), quantitative real-time PCR, enzyme-linked immunosorbent assay, and Western blot analysis were performed to verify the interference effects. As a result, a recombinant adenovirus was successfully constructed and effectively packaged in AD293 cells, and it significantly inhibited HBs mRNA and protein expression in vitro. Our study may provide a novel tool to study HBs function.

Protein S.

Protein S (PS) is a vitamin K-dependent plasma glycoprotein. Around 60-70% of PS in plasma is noncovalently bound to C4-binding protein (C4BP). Free PS functions as a cofactor that enhances the activity of activated protein C (APC) in the proteolytic degradation of activated factors V and VIII. PS also has a more recently described APC-independent ability to directly inhibit prothrombinase and tenase by direct binding of activated factors V, VIII, and X. Given that PS is one of the major naturally occurring inhibitors of coagulation, acquired or hereditary deficiencies of this protein result in excessive thrombin generation. As a vast array of mutations are responsible for hereditary PS deficiencies, screening for their presence by DNA testing would require sequencing each entire gene involving numerous exons. Moreover, the knowledge of the gene mutation does not offer any benefit in the treatment of thrombophilic families, so the routine molecular characterization is not indicative. These defects are detected by functional or immunological assays for free and total PS forms. Given that functional PS assays may detect some forms of PS deficiency that free PS immunoassays may miss, it is recommended to include them for initial testing along with immunoassays for free PS, although they should be used with caution. Functional PS assays are subject to multiple interference. For example in the presence of lupus anticoagulant (LA), only free PS immunoassays are recommended for initial testing. PS antigen assays are more popular with most laboratories.

Distúrbios de coagulação em pacientes com osteonecrose da cabeça femoral / Coagulation disorders in patients with femoral head osteonecrosis

Objetivo: Comparar a ocorrência de trombofilias em pacientes com osteonecrose idiopática da cabeça femoral em relação aos pacientes com osteonecrose secundária da cabeça femoral. Métodos: Um total de 24 pacientes consecutivos foram avaliados, sendo oito portadores de osteonecrose idiopática e 16 de osteonecrose secundária. Os exames realizados na detecção de trombofilias foram as dosagens de proteína C, proteína S e antitrombina e as pesquisas de mutações nos genes da protrombina e do fator V. Comparamos estatisticamente os resultados através do cálculo da razão de chances ou odds ratio das diferentes trombofilias entre os dois grupos. Resultados: O odds ratio para a deficiência da proteína S e deficiência da proteína C entre os grupos idiopático e secundário foram respectivamente 5 e 2,14. Desta maneira, um indivíduo com osteonecrose idiopática possui uma chance 5 vezes maior de apresentar deficiência da proteína S e 2,14 vezes maior de apresentar deficiência da proteína C do que um indivíduo com osteonecrose secundária. Conclusão: Pacientes com osteonecrose idiopática têm maiores chances de apresentar trombofilias do que aqueles com osteonecrose secundária, sugerindo que estes distúrbios de coagulação podem desempenhar um papel importante na patogênese dos casos de osteonecrose onde não há inicialmente nenhum fator de risco identificável. Nível de Evidência III, Estudo de Caso-Controle.

Objective: To compare the occurrence of thrombophilic disorders in patients with idiopathic osteonecrosis of the femoral head and patients with secondary osteonecrosis of the femoral head. Methods: Twenty-four consecutive patients were enrolled, with eight of them presenting idiopathic osteonecrosis and 16 presenting secondary osteonecrosis. The tests for detection of thrombophilic disorders were measurements of protein C, protein S and antithrombin levels and detection of prothrombin and factor V gene mutations. We compared the results using the odds ratio statistics for the thrombophilic disorders between the two groups. Results: The odds ratio for the protein S deficiency and protein C deficiency between the idiopathic and secondary groups were 5 and 2.14, respectively. Thus, an individual with idiopathic os teonecrosis has 5 times more chance of presenting protein S deficiency and 2.14 times more chance of presenting protein C deficiency than an individual with secondary osteonecrosis. Conclusion: Patients with idiopathic osteonecrosis have more chances of presenting thrombophilias than those with secondary osteonecrosis, suggesting these coagulation disorders can play an important role in the pathogenesis of the osteonecrosis in cases where there was no initial risk factor recognized. Level of Evidence III, Case-Control Study.

Clonal analysis of hepatitis B viruses among blood donors from Joinville, Brazil: evidence of dual infections, intragenotype recombination and markers of risk for hepatocellular carcinoma.

Hepatitis B virus (HBV) is classified into seven major genotypes (A-H). Brazil, a country of continental proportions, has three prevailing lineages of HBV genotypes A, D, and F. Distinct HBV genotypes have been associated with differential risk of disease progression. Pre-S gene deletions and single nucleotide polymorphisms have also been linked to progression to liver diseases. In this study, the molecular epidemiology of HBV was examined in Southern Brazil. The occurrence of multiple HBV infections, HBV recombination, and genetic markers of disease progression were also evaluated. Seventy-eight persons infected with HBV had their viruses characterized molecularly by nested PCR, DNA sequencing, and phylogenetic inference. Multiple infections and recombinant viruses were evaluated by clonal and bootscanning analyses. The vast majority (96%) of the strains belonged to different D subgenotypes. Three of the four strains with unresolved genotypic classification showed evidence of dual infections with distinct D subgenotypes by clonal analysis. There was also evidence of intragenotype mosaic viruses. While four viruses had pre-S deletions as major variants, another two displayed minor variants with such characteristics. One strain carried the F141L mutation, associated recently with increased risk of hepatocellular carcinoma. These results emphasize the need for monitoring HBV genotype distribution around South America, as well as for the presence of genetic markers of disease progression in subjects diagnosed with HBV recently.

Prevalence of common thrombophilia markers and risk factors in Indian patients with primary venous thrombosis.

CONTEXT AND OBJECTIVE: venous thrombosis occurs as a result of interaction of genetic and acquired factors including activated protein C resistance (APC-R), fibrinogen levels, antithrombin, protein C, protein S, lupus anticoagulants and anticardiolipin antibodies. This study was aimed at determining the prevalence of these common thrombophilia markers in Asian Indians with primary venous thrombosis. DESIGN AND SETTING: this was a cross-sectional study carried out in Mumbai. METHODS: samples from 78 patients with a confirmed diagnosis of venous thrombosis and 50 controls were tested. Semi-quantitative estimation (functional assays) of protein C, protein S and antithrombin was performed. Quantitative estimation of fibrinogen was done using the Clauss method. Lupus anticoagulants were screened using lupus-sensitive activated partial thromboplastin time and ß2-glycoprotein-I dependent anticardiolipin antibodies were estimated by ELISA. APC-R was measured using a clotting-based method with factor V deficient plasma and Crotalus viridis venom. Statistical analysis was performed using Epi-info (version 6). RESULTS: the popliteal vein was the most commonly involved site. Forty-four samples (56%) gave abnormal results. The commonest were elevated fibrinogen and APC-R (17.9% each), followed by low protein S (16.6%). CONCLUSIONS: this study confirms the literature findings that fibrinogen level estimation and screening for APC-R are important for the work-up on venous thrombosis patients since these, singly or in combination, may lead to a primary thrombotic episode. The frequency of the other thrombophilia markers was higher among the patients than among the controls, but without statistically significant difference.