Javier García-Frade

Experimental and Clinical Regenerative Capability of Human Bone Marrow Cells After Myocardial Infarction

Bone marrow mononuclear cells (BMCs) from 20 patients with extensive reperfused myocardial infarction (MI) were used to assess their myocardial regenerative capability “in vitro” and their effect on postinfarction left ventricular (LV) remodeling. Human BMCs were labeled, seeded on top of cryoinjured mice heart slices, and cultured. BMCs showed tropism for and ability to graft into the damaged mouse cardiac tissue and, after 1 week, acquired a cardiomyocyte phenotype and expressed cardiac proteins, including connexin43. In the clinical trial, autologous BMCs (78±41×106 per patient) were intracoronarily transplanted 13.5±5.5 days after MI. There were no adverse effects on microvascular function or myocardial injury. No major cardiac events occurred up to 11±5 months. At 6 months, magnetic resonance showed a decrease in the end-systolic volume, improvement of regional and global LV function, and increased thickness of the infarcted wall, whereas coronary restenosis was only 15%. No changes were found in a nonrandomized contemporary control group. Thus, BMCs are capable of nesting into the damaged myocardium and acquire a cardiac cell phenotype in vitro as well as safely benefiting ventricular remodeling in vivo. Large-scale randomized trials are needed now to assess the clinical efficacy of this treatment.

Successful discontinuation of eltrombopag after complete remission in patients with primary immune thrombocytopenia

Eltrombopag is effective and safe in immune thrombocytopenia (ITP). Some patients may sustain their platelet response when treatment is withdrawn but the frequency of this phenomenon is unknown. We retrospectively evaluated 260 adult primary ITP patients (165 women and 95 men; median age, 62 years) treated with eltrombopag after a median time from diagnosis of 24 months. Among the 201 patients who achieved a complete remission (platelet count >100 × 109/l), eltrombopag was discontinued in 80 patients. Reasons for eltrombopag discontinuation were: persistent response despite a reduction in dose over time (n = 33), platelet count >400 × 109/l (n = 29), patients request (n = 5), elevated aspartate aminotransferase (n = 3), diarrhea (n = 3), thrombosis (n = 3), and other reasons (n = 4). Of the 49 evaluable patients, 26 patients showed sustained response after discontinuing eltrombopag without additional ITP therapy, with a median follow‐up of 9 (range, 6–25) months. These patients were characterized by a median time since ITP diagnosis of 46.5 months, with 4/26 having ITP < 1 year. Eleven patients were male and their median age was 59 years. They received a median of 4 previous treatment lines and 42% were splenectomized. No predictive factors of sustained response after eltrombopag withdrawal were identified. Platelet response following eltrombopag cessation may be sustained in an important percentage of adult primary ITP patients who achieved CR with eltrombopag. However, reliable markers for predicting which patients will have this response are needed. Am. J. Hematol. 90:E40–E43, 2015.

Use of eltrombopag after romiplostim in primary immune thrombocytopenia

The thrombopoietin receptor agonists (THPO‐RAs), romiplostim and eltrombopag, are effective and safe in immune thrombocytopenia (ITP). However, the value of their sequential use when no response is achieved or when adverse events occur with one THPO‐RA has not been clearly established. Here we retrospectively evaluated 51 primary ITP adult patients treated with romiplostim followed by eltrombopag. The median age of our cohort was 49 (range, 18–83) years. There were 32 women and 19 men. The median duration of romiplostim use before switching to eltrombopag was 12 (interquartile range 5–21) months. The reasons for switching were: lack of efficacy (n = 25), patient preference (n = 16), platelet‐count fluctuation (n = 6) and side‐effects (n = 4). The response rate to eltrombopag was 80% (41/51), including 67% (n = 35) complete responses. After a median follow‐up of 14 months, 31 patients maintained their response. Efficacy was maintained after switching in all patients in the patient preference, platelet‐count fluctuation and side‐effect groups. 33% of patients experienced one or more adverse events during treatment with eltrombopag. We consider the use of eltrombopag after romiplostim for treating ITP to be effective and safe. Response to eltrombopag was related to the cause of romiplostim discontinuation.

Molecular and clinical profile of von Willebrand disease in Spain (PCM–EVW–ES): Proposal for a new diagnostic paradigm

The diagnosis of von Willebrand disease (VWD) remains difficult in a significant proportion of patients. A Spanish multicentre study investigated a cohort of 556 patients from 330 families who were analysed centrally. VWD was confirmed in 480. Next generation sequencing (NGS) of the whole coding VWF was carried out in all recruited patients, compared with the phenotype, and a final diagnosis established. A total of 238 different VWF mutations were found, 154 were not included in the Leiden Open Variation Database (LOVD). Of the patients, 463 were found to have VWF mutation/s. A good phenotypic/genotypic association was estimated in 96.5% of the patients. One hundred seventy-four patients had two or more mutations. Occasionally a predominant phenotype masked the presence of a second abnormality. One hundred sixteen patients presented with mutations that had previously been associated with increased von Willebrand factor (VWF) clearance. RIPA unavailability, central phenotypic results disagreement and difficult distinction between severe type 1 and type 3 VWD prevented a clear diagnosis in 70 patients. The NGS study facilitated an appropriate classification in 63 of them. The remaining seven patients presented with a VWF novel mutation pending further investigation. In five patients with a type 3 and two with a type 2A or 2B phenotype with no mutation, an acquired von Willebrand syndrome (AVWS) was suspected/confirmed. These data seem to support NGS as a first line efficient and faster paradigm in VWD diagnosis.

Absence of accelerated atherosclerotic disease progression after intracoronary infusion of bone marrow derived mononuclear cells in patients with acute myocardial infarction--angiographic and intravascular ultrasound--results from the TErapia Celular Aplicada al Miocardio Pilot study.

BACKGROUND We tried to evaluate a putative negative effect on coronary atherosclerosis in patients receiving intracoronary infusion of unfractionated bone marrow mononuclear cells (BMMC) following an acute ST-elevation myocardial infarction. Peripheral blood mononuclear cells or enriched CD133(+) BMMC have been associated with accelerated atherosclerosis of the distal segment of the infarct related artery (IRA). METHODS Thirty-seven patients with ST-elevation myocardial infarction from the TECAM pilot study underwent intracoronary infusion of autologous BMMC 9 +/- 3.1 days after onset of symptoms. We compared angiographic changes from baseline to 9 months of follow-up in the distal non-stented segment of the IRA, as well as in the contralateral coronary artery, with a matched control group. A subgroup of 15 treated patients underwent additional IVUS within the distal segment of the IRA. RESULTS No difference between stem cell and control group were found regarding changes in minimum lumen diameter (0.006 +/- 0.42 vs 0.06 +/- 0.41 mm, P = ns) and the percentage of stenosis (-2.68 +/- 12.33% vs -1.78 +/- 8.75%, P = ns) at follow-up. Likewise, no differences were seen regarding changes in the contralateral artery (minimum lumen diameter -0.004 +/- 0.54 mm vs -0.06 +/- 0.35 mm, P = ns). In the intravascular ultrasound substudy, no changes were demonstrated comparing baseline versus follow-up in maximum area stenosis and plaque volume. CONCLUSIONS In this pilot study, analysis of a subgroup of patients found that intracoronary injection of unfractionated BMMC in patients with acute ST-elevation myocardial infarction was not associated with accelerated atherosclerosis progression at mid term. Prospective, randomised studies in large cohorts with long-term angiographic and intravascular ultrasound follow-up are necessary to determine the safety of this therapy.

Comparison of Neointimal Hyperplasia With Drug-Eluting Stents Versus Bare Metal Stents in Patients Undergoing Intracoronary Bone-Marrow Mononuclear Cell Transplantation Following Acute Myocardial Infarction

The aims of this study were to assess the safety of drug-eluting stent (DES) use and to compare the incidence of in-stent restenosis (ISR) and neointimal hyperplasia formation according to the type of stent implanted (DES vs bare-metal stents [BMS]) in patients who underwent intracoronary bone marrow mononuclear cell transplantation after acute ST elevation myocardial infarction. Fifty-nine patients with successfully revascularized ST elevation myocardial infarction (37 using BMS and 22 using DES) underwent paired angiographic examinations at baseline and 6 to 9 months after the intracoronary injection of 91 million +/- 56 million autologous bone marrow mononuclear cells. A subgroup of 30 patients also underwent serial intravascular ultrasound examinations. Off-line angiographic assessment showed 4 cases of binary ISR, primarily in BMS (3 cases), and no major adverse cardiac events were associated with stent type (mean follow-up period 41 +/- 10 months). At follow-up, angiographic late luminal loss was significantly lower in patients with DES than in those patients with BMS (0.35 +/- 0.66 vs 0.71 +/- 0.38 mm, p = 0.011). Multivariate analysis identified the use of DES (beta = -0.32, 95% confidence interval [CI] -0.57 to -0.26, p = 0.03) and a smaller baseline reference vessel diameter (beta = 0.29, 95% CI 0.04 to 0.54, p = 0.02) as independent predictors of lower late loss. Moreover, intravascular ultrasound showed a significant reduction of in-stent neointimal hyperplasia formation related to DES use compared with BMS use (Delta neointimal hyperplasia volume 5.4 mm(3) [95% CI 2.7 to 28.1] vs 35.9 mm(3) [95% CI 22.0 to 43.6], p = 0.035). In conclusion, these findings suggest that the use of DES is safe and may prevent ISR and neointimal hyperplasia formation in patients who undergo intracoronary bone marrow mononuclear cell transplantation after a successfully revascularized ST elevation myocardial infarction.

Molecular and clinical profile of von Willebrand disease in Spain (PCM-EVW-ES): Comprehensive genetic analysis by next-generation sequencing of 480 patients

Molecular diagnosis of patients with von Willebrand disease is pending in most populations due to the complexity and high cost of conventional molecular analyses. The need for molecular and clinical characterization of von Willebrand disease in Spain prompted the creation of a multicenter project (PCM-EVW-ES) that resulted in the largest prospective cohort study of patients with all types of von Willebrand disease. Molecular analysis of relevant regions of the VWF, including intronic and promoter regions, was achieved in the 556 individuals recruited via the development of a simple, innovative, relatively low-cost protocol based on microfluidic technology and next-generation sequencing. A total of 704 variants (237 different) were identified along VWF, 155 of which had not been previously recorded in the international mutation database. The potential pathogenic effect of these variants was assessed by in silico analysis. Furthermore, four short tandem repeats were analyzed in order to evaluate the ancestral origin of recurrent mutations. The outcome of genetic analysis allowed for the reclassification of 110 patients, identification of 37 asymptomatic carriers (important for genetic counseling) and re-inclusion of 43 patients previously excluded by phenotyping results. In total, 480 patients were definitively diagnosed. Candidate mutations were identified in all patients except 13 type 1 von Willebrand disease, yielding a high genotype-phenotype correlation. Our data reinforce the capital importance and usefulness of genetics in von Willebrand disease diagnostics. The progressive implementation of molecular study as the first-line test for routine diagnosis of this condition will lead to increasingly more personalized and effective care for this patient population.

Cirrosis biliar primaria, síndrome "sicca" y anemia hemolítica autoinmune

La cirrosis biliar primaria (CBP) es un proceso colestático que puede asociarse a diferentes manifestaciones autoinmunes tales como síndrome “sicca”, artritis reumatoidea, esclerodermia, síndrome CREST, fenómeno de Raynaud, enfermedades tiroideas, anemia perniciosa, etc. (1,2). La anemia hemolítica autoinmune (AHAI) es un proceso de etiología diversa comúnmente idiopático, que se ha descrito con escasa frecuencia en asociación con la CBP (3-7).

Unraveling the effect of silent, intronic and missense mutations on VWF splicing: contribution of next generation sequencing in the study of mRNA.

Large studies in von Willebrand disease patients, including Spanish and Portuguese registries, led to the identification of >250 different mutations. It is a challenge to determine the pathogenic effect of potential splice site mutations on VWF mRNA. This study aimed to elucidate the true effects of 18 mutations on VWF mRNA processing, investigate the contribution of next-generation sequencing to in vivo mRNA study in von Willebrand disease, and compare the findings with in silico prediction. RNA extracted from patient platelets and leukocytes was amplified by RT-PCR and sequenced using Sanger and next generation sequencing techniques. Eight mutations affected VWF splicing: c.1533+1G>A, c.5664+2T>C and c.546G>A (p.=) prompted exon skipping; c.3223-7_3236dup and c.7082-2A>G resulted in activation of cryptic sites; c.3379+1G>A and c.7437G>A) demonstrated both molecular pathogenic mechanisms simultaneously; and the p.Cys370Tyr missense mutation generated two aberrant transcripts. Of note, the complete effect of three mutations was provided by next generation sequencing alone because of low expression of the aberrant transcripts. In the remaining 10 mutations, no effect was elucidated in the experiments. However, the differential findings obtained in platelets and leukocytes provided substantial evidence that four of these would have an effect on VWF levels. In this first report using next generation sequencing technology to unravel the effects of VWF mutations on splicing, the technique yielded valuable information. Our data bring to light the importance of studying the effect of synonymous and missense mutations on VWF splicing to improve the current knowledge of the molecular mechanisms behind von Willebrand disease. clinicaltrials.gov identifier:02869074.