Defenestrated endothelium delays liver-directed gene transfer in hemophilia A mice.

Hemophilia A is an inherited bleeding disorder caused by defective or deficient coagulation factor VIII (FVIII) activity. Until recently, the only treatment for prevention of bleeding involved IV administration of FVIII. Gene therapy with adeno-associated vectors (AAVs) has shown some efficacy in patients with hemophilia A. However, limitations persist due to AAV-induced cellular stress, immunogenicity, and reduced durability of gene expression. Herein, we examined the efficacy of liver-directed gene transfer in FVIII knock-out mice by AAV8-GFP. Surprisingly, compared with control mice, FVIII knockout (F8TKO) mice showed significant delay in AAV8-GFP transfer in the liver. We found that the delay in liver-directed gene transfer in F8TKO mice was associated with absence of liver sinusoidal endothelial cell (LSEC) fenestration, which led to aberrant expression of several sinusoidal endothelial proteins, causing increased capillarization and decreased permeability of LSECs. This is the first study to link impaired liver-directed gene transfer to liver-endothelium maladaptive structural changes associated with FVIII deficiency in mice.

Activated protein C has a regulatory role in factor VIII function.

Mechanisms thought to regulate activated factor VIII (FVIIIa) cofactor function include A2-domain dissociation and activated protein C (APC) cleavage. Unlike A2-domain dissociation, there is no known phenotype associated with altered APC cleavage of FVIII, and biochemical studies have suggested APC plays a marginal role in FVIIIa regulation. However, the in vivo contribution of FVIIIa inactivation by APC is unexplored. Here we compared wild-type B-domainless FVIII (FVIII-WT) recombinant protein with an APC-resistant FVIII variant (FVIII-R336Q/R562Q; FVIII-QQ). FVIII-QQ demonstrated expected APC resistance without other changes in procoagulant function or A2-domain dissociation. In plasma-based studies, FVIII-WT/FVIIIa-WT demonstrated dose-dependent sensitivity to APC with or without protein S, whereas FVIII-QQ/FVIIIa-QQ did not. Importantly, FVIII-QQ demonstrated approximately fivefold increased procoagulant function relative to FVIII-WT in the tail clip and ferric chloride injury models in hemophilia A (HA) mice. To minimize the contribution of FV inactivation by APC in vivo, a tail clip assay was performed in homozygous HA/FV Leiden (FVL) mice infused with FVIII-QQ or FVIII-WT in the presence or absence of monoclonal antibody 1609, an antibody that blocks murine PC/APC hemostatic function. FVIII-QQ again demonstrated enhanced hemostatic function in HA/FVL mice; however, FVIII-QQ and FVIII-WT performed analogously in the presence of the PC/APC inhibitory antibody, indicating the increased hemostatic effect of FVIII-QQ was APC specific. Our data demonstrate APC contributes to the in vivo regulation of FVIIIa, which has the potential to be exploited to develop novel HA therapeutics.

Dilataciones endoscópicas del tracto gastrointestinal / Endoscopic dilation of the gastrointestinal tract

Las dilataciones en el tracto gastrointestinal se llevan a cabo para aliviar la obstrucción sintomática, ya sea funcional u orgánica, secundarias a una variedad de patologías tanto benignas como malignas. Con el advenimiento de las nuevas tecnologías, virtualmente toda estenosis digestiva puede ser manejada en forma mínimamente invasiva. Pese a su amplia difusión en la práctica actual, existen pocos estudios controlados que comparen las diferentes modalidades de dilatación. En el presente artículo realizamos una revisión de esta técnica, así como de la evidencia disponible para su aplicación en los diferentes segmentos del tracto gastrointestinal. El futuro de la dilatación incluye el desarrollo de dilatadores que permitan evaluar la dilatación durante su realización. Estos advenimientos, así como la ejecución de estudios controlados prospectivos van a mejorar las indicaciones, beneficios y riesgos para cada uno de los sistemas de dilatación existentes.

The endoscopic dilation of the gastrointestinal tract is carried out to relieve either functional or organic disorders, secondary to a variety of both benign and malignant diseases. With the advent of new technologies, virtually all digestive stenosis can be managed in a minimally invasive way. Despite its wide dissemination in actual practice, there are few controlled studies comparing the different forms of endoscopic dilation. In this article, we review this technique and the evidence available for application in different segments of the gastrointestinal tract. The future of the dilations includes the development of dilators to assess dilation during the procedure. These advents and the implementation of prospective controlled studies will improve the indications, benefits and risks for each of the existing systems of dilations.

Cytokine profile and FVIII inhibitors development in haemophilia A.

Haemophilia A is a hereditary bleeding disorder linked to the X chromosome characterized by a deficiency or defect in the coagulation factor VIII (FVIII). Individuals with this coagulopathy require constant infusions of FVIII to maintain their physical integrity and haemostasis. During treatment, some patients develop an immune response that produces antibodies to FVIII, also called inhibitors, affecting the pro-coagulant activity of this protein. Despite the clinical relevance of FVIII inhibitors, the immune mechanisms that lead to their production are not known. This study investigated the immunological cytokine profile using plasma from HA patients which were either positive or negative for FVIII inhibitors and from healthy individuals. The results showed that healthy individuals and HA patients that do not develop FVIII inhibitors have a mixed immune response profile with high secretion of IFN-γ, TNF-α IL-2 and IL-5. In contrast, HA patients with FVIII inhibitors exhibited an anti-inflammatory/regulatory immune response characterized by low levels of all measured cytokines except for IL-4 and IL-10. This profile may be related to the development and maintenance of the FVIII inhibitors. By comparing the cytokine profiles of the three different groups we have established a model explaining the immune activation resulting in the production of FVIII inhibitors in haemophilia A patients.

Hemofilia II. Aspectos moleculares y de genética poblacional / Hemophilia II. Molecular and population genetics

La prevalencia de la hemofilia esporádica fue estimada hace más de 40 años y se demostró que aproximadamente un tercio de los casos son de novo. La mayoría de las mutaciones que ocurren en la hemofilia se producen durante la espermatogénesis masculina; en otros casos, los cambios ocurren en los estadios tempranos de desarrollo del embrión o una mutación germinal en la madre. El proceso de inactivación del cromosoma X es al azar. Extensos estudios han evidenciado que son más frecuentes las mutaciones en las meiosis masculinas que en las femeninas, con una proporción global de 3,5/1, especialmente las inversiones de los intrones 22 y 1. Se revisaron aspectos moleculares y bioquímicos de los factores VIII y IX. Destacamos la importancia del dominio B del factor VIII que contribuye a múltiples funciones esenciales, como el control de la calidad de la síntesis, la secreción, la unión con los fosfolípidos plaquetarios, la inactivación y el aclaramiento de la molécula completa

The sporadic hemophilia prevalence was estimated more than 40 years ago and it was shown that approximately a third of the cases are novo. Most of the mutations that occur in hemophilia are produced during the male spermatogenesis; in other cases, they occur in early stages of the embrión development or in the mother a germinal mutation. The X-cromosoma- inactivation process is at random. Extended studies have shown that male meiosis are more frequent than female ones, with a global proportion of 3,5/1, specially introns inversions 22 and 1. There were revised molecular and biochemical aspects of factors VIII and IX. We ruled out the importance of B domain in factor VIII, which contributes to multiple essential functions, as the quality control of synthesis, secretion, union with platelet phospholipids, inactivation and complete clearance of the molecule

Severe haemophilia A patients have reduced numbers of peripheral memory B cells.

The development of inhibitors is a complication of replacement treatment in Haemophilia. Loss of factor VIII-specific memory B cells in the spleen is associated with down regulation of antibodies in mice treated with high doses of FVIII, but changes in B cell memory have not been described in haemophilic patients. The aim of this study was to evaluate the phenotype of circulating lymphocytes in severe haemophilia A. Twenty patients with inhibitors (PI), 22 without inhibitors (P), nine patients during immune tolerance induction (ITI) treatment and 20 healthy donors (HD) were included. Peripheral blood lymphocytes were examined using flow cytometry. Anti-FVIII antibodies were measured using Bethesda and flow cytometry. Percentages of T subsets and B lymphocytes were similar in all groups. In contrast, memory B cells (CD27+) were decreased in PI and P compared with HD, but the level of significance was higher in PI (P = 0.001) than P (P = 0.01). PI with high level of anti-FVIII antibodies presented the lowest B memory values. CD70 expression was also lowest in PI. Non-switched CD27+ subpopulation (IgD+) was prevalent in PI, but did not show statistical significance. When ITI failed, the percentages of CD27+ B cells after 12 months of ITI were lowest. In a longitudinal study performed in four patients, an increased percentage of CD27+ and CD70+ B cells during ITI was found. This work suggests that different peripheral lymphocyte markers, such as CD27 and CD70 on B cells, may be helpful to evaluate anti-FVIII response and to monitor the success of ITI.

Evaluation of the host response to endotoxemia of FVIII and FIX deficient mice.

For several years, coagulation has been implicated in the pathogenesis of sepsis. However, results from clinical trials with natural anticoagulants, as well as studies with knock-out mice for specific coagulation factors yielded conflicting results on the role of coagulation in the pathogenesis of sepsis. The aim of this study was to evaluate the impact of severe The factor VIII:C (FVIII:C) and factor IX:C (FIX:C) deficiency on a lipopolysaccharide (LPS)-induced murine model of sepsis. FVIII:C and FIX:C deficient mice, and their haemostatic normal littermate controls were challenged with LPS, and several parameters of the host response were evaluated: seven-day survival experiments were performed using two dose levels of LPS; biochemical and histological markers of tissue damage, coagulation parameters, and pro-inflammatory cytokines were evaluated at baseline and after 3 h and 6 h after an injection of LPS. Severe FVIII and FIX deficiency were compatible with normal survival in experimental sepsis. In addition, LPS-induced tissue damage and coagulation activation were similar in FVIII or FIX deficient mice compared to their respective controls. A lower release of pro-inflammatory cytokines was observed in FIX but not in FVIII deficient mice. Severe FIX or FVIII deficiency does not protect mice from mortality or from tissue damage in the endotoxemia model, supporting the hypothesis that FVIII and FIX are not critical to the pathogenesis of experimental sepsis.

[Fibrinolytic activity of gingival fluid in hemophiliac patients]. / Actividad fibrinolítica del fluído gingival en pacientes hemofílicos.

The purpose of this investigation is to compare the fibrinolytic activity in the gingival fluid, of 32 hemophilic patients with 36 normal subjects. Oral examination was carried out in both groups in order to determine the extent of periodontal disease, using the Ramfjord Periodontal Index. The disease was classified in three grades according to the depth of the gingival crevices: grade I: 0 to 3 mm (Normal Deepness); grade II: from 3 to 6 mm; grade III higher than 6 mm. The fibrinolytic activity was measured in fibrin plates, prepared with thrombin and fibrinogen with and without plasminogen. It was found that the fibrinolytic activity was similar in the groups studied and related to the depth of the gingival crevices, with the exception of hemophilic patients with grade I disease, who showed higher fibrinolytic activity in the fibrin plates with plasminogen, than in the corresponding controls (p less than 0.001). This result could possibly be due to mucosal traumatism during the withdrawal of the sample, which permitted the release of plasminogen activators from the damaged vessels. It is important to mention the absence of grade III periodontal disease in the hemophilic group. The reason for this result could be due to the important bleeding occurring in grade II, which induces the patient to seek immediate professional attention. The above mentioned results suggest that the fibrinolytic activity of the gingival fluid in hemophilic and control patients is directly related to the degree of periodontal disease.