Abstract
Neonatal platelets are hypo-reactive to the tyrosine kinase-linked receptor agonist collagen. Here, we have investigated whether the hypo-responsiveness is related to altered levels of ...glycoprotein VI (GPVI) and integrin α2β1, or to defects in downstream signalling events by comparison to platelet activation by C-type lectin-like receptor 2 (CLEC-2). GPVI and CLEC-2 activate a Src- and Syk-dependent signalling pathway upstream of phospholipase C (PLC) γ2. Phosphorylation of a conserved YxxL sequence known as a (hemi) immunotyrosine-based-activation-motif (ITAM) in both receptors is critical for Syk activation. Platelets from human pre-term and full-term neonates display mildly reduced expression of GPVI and CLEC-2, as well as integrin αIIbβ3, accounted for at the transcriptional level. They are also hypo-responsive to the two ITAM receptors, as shown by measurement of integrin αIIbβ3 activation, P-selectin expression and Syk and PLCγ2 phosphorylation. Mouse platelets are also hypo-responsive to GPVI and CLEC-2 from late gestation to 2 weeks of age, as determined by measurement of integrin αIIbβ3 activation. In contrast, the response to G protein-coupled receptor agonists was only mildly reduced and in some cases not altered in neonatal platelets of both species. A reduction in response to GPVI and CLEC-2, but not protease-activated receptor 4 (PAR-4) peptide, was also observed in adult mouse platelets following immune thrombocytopenia, whereas receptor expression was not impaired. Our results demonstrate developmental differences in platelet responsiveness to GPVI and CLEC-2, and also following immune platelet depletion leading to reduced Syk activation. The rapid generation of platelets during development or following platelet depletion is achieved at the expense of signalling by ITAM-coupled receptors.
Chronic myelomonocytic leukemia (CMML) is frequently associated with mutations in the rat sarcoma gene (RAS), leading to worse prognosis. RAS mutations result in active RAS-GTP proteins, favoring ...myeloid cell proliferation and survival and inducing the NLRP3 inflammasome together with the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), which promote caspase-1 activation and interleukin (IL)-1β release. Here, we report, in a cohort of CMML patients with mutations in KRAS, a constitutive activation of the NLRP3 inflammasome in monocytes, evidenced by ASC oligomerization and IL-1β release, as well as a specific inflammatory cytokine signature. Treatment of a CMML patient with a KRAS
mutation using the IL-1 receptor blocker anakinra inhibits NLRP3 inflammasome activation, reduces monocyte count, and improves the patient's clinical status, enabling a stem cell transplant. This reveals a basal inflammasome activation in RAS-mutated CMML patients and suggests potential therapeutic applications of NLRP3 and IL-1 blockers.
Background
Plasma exchange (PLEX) is a therapeutic option in the treatment of acute attacks of Demyelinating Diseases of the Central Nervous System (DDCNS). Factors related with PLEX response are not ...well established.
Methods
Descriptive and retrospective study. We included patients treated with PLEX for acute attacks of DDCNS between 2008 and 2017. We recorded demographics, clinical and treatment-related data, and Expanded Disability Status Scale (EDSS) score at admission, at discharge, and at 6 months.
Results
We included 64 patients. Forty-eight (75%) were female with a mean age of 48.28 ± 11.5 years. Half of our patients were diagnosed with multiple sclerosis. Clinical improvement was achieved in 51.6% at discharge and 62.5% at 6 months. The logistic regression model showed that EDSS score > 3 at admission (
p
= 0.04) and early clinical improvement with PLEX (
p
= 0.00) were predictors of good response to PLEX at discharge and at 6 months, respectively. No serious adverse effects were identified.
Conclusions
PLEX is a safe and effective treatment for acute attacks of DDCNS. EDSS score at admission and early clinical improvement with PLEX were factors associated with good response to PLEX.
BACKGROUNDPatientsʼ experience with health care is becoming a key component for the provision of a patient-centered health care model. The aim of this study was to assess the experience with health ...care of patients with inflammatory arthritis and patient- and health care–related factors.
METHODSPatients responded to an anonymous survey provided by their treating clinical teams. The survey comprised the validated 12-item IEXPAC (Instrument to Evaluate the EXperience of PAtients with Chronic diseases) tool and demographic variables and health care–related characteristics that may affect patientsʼ experience.
RESULTSA total of 359 of 625 surveys were returned (response rate, 57.4%). Overall, patient responses were positive (>60% gave “always/mostly” answers) for statements assessing the interaction between patients and health care professionals or patient self-management following health care professional guidance. However, positive patient responses for items regarding patient interaction with the health care system via the internet or with other patients were less than 13%. Only 25.6% of patients who had been hospitalized reported receiving a follow-up call or visit following discharge. In the bivariate analysis, experience scores were higher (better experience) in men, those seen by fewer specialists or by the same physician, and in patients treated with a fewer number of drugs or with subcutaneous/intravenous drugs. Multivariate analyses identified regular follow-up by the same physician and treatment with subcutaneous/intravenous drugs as variables associated with a better patient experience.
CONCLUSIONSThis study identified areas of care for patients with inflammatory arthritis with the potential to improve patientsʼ experience and highlights the importance of patient-physician relationships and comprehensive patient care.
The regulatory role of microRNAs (miRNAs) is mainly mediated by their effect on protein expression and is recognized in a multitude of pathophysiological processes. In recent decades, accumulating ...evidence has interest in these factors as modulatory elements of cardiovascular pathophysiology. Furthermore, additional biological processes have been identified as new components of cardiovascular disease etiology. In particular, inflammation is now considered an important cardiovascular risk factor. Thus, in the present review, we will focus on the role of a subset of miRNAs called inflamma-miRs that may regulate inflammatory status in the development of cardiovascular pathology. According to published data, the most representative candidates that play functional roles in thromboinflammation are miR-21, miR-33, miR-34a, miR-146a, miR-155, and miR-223. We will describe the functions of these miRNAs in several cardiovascular pathologies in depth, with specific emphasis on the molecular mechanisms related to atherogenesis. We will also discuss the latest findings on the role of miRNAs as regulators of neutrophil extracellular traps and their impact on cardiovascular diseases. Overall, the data suggest that the use of miRNAs as therapeutic tools or biomarkers may improve the diagnosis or prognosis of adverse cardiovascular events in inflammatory diseases. Thus, targeting or increasing the levels of adequate inflamma-miRs at different stages of disease could help mitigate or avoid the development of cardiovascular morbidities.
Background and objectives: Glycerol phenylbutyrate (GPB) has demonstrated safety and efficacy in patients with urea cycle disorders (UCDs) by means of its clinical trial program, but there are ...limited data in clinical practice. In order to analyze the efficacy and safety of GPB in clinical practice, here we present a national Spanish experience after direct switching from another nitrogen scavenger to GPB. Methods: This observational, retrospective, multicenter study was performed in 48 UCD patients (age 11.7 ± 8.2 years) switching to GPB in 13 centers from nine Spanish regions. Clinical, biochemical, and nutritional data were collected at three different times: prior to GPB introduction, at first follow-up assessment, and after one year of GPB treatment. Number of related adverse effects and hyperammonemic crisis 12 months before and after GPB introduction were recorded. Results: GPB was administered at a 247.8 ± 102.1 mg/kg/day dose, compared to 262.6 ± 126.1 mg/kg/day of previous scavenger (46/48 Na-phenylbutyrate). At first follow-up (79 ± 59 days), a statistically significant reduction in ammonia (from 40.2 ± 17.3 to 32.6 ± 13.9 μmol/L, p < 0.001) and glutamine levels (from 791.4 ± 289.8 to 648.6 ± 247.41 μmol/L, p < 0.001) was observed. After one year of GPB treatment (411 ± 92 days), we observed an improved metabolic control (maintenance of ammonia and glutamine reduction, with improved branched chain amino acids profile), and a reduction in hyperammonemic crisis rate (from 0.3 ± 0.7 to less than 0.1 ± 0.3 crisis/patients/year, p = 0.02) and related adverse effects (RAE, from 0.5 to less than 0.1 RAEs/patients/year p < 0.001). Conclusions: This study demonstrates the safety of direct switching from other nitrogen scavengers to GPB in clinical practice, which improves efficacy, metabolic control, and RAE compared to previous treatments.
Factors associated with chronic heart failure (CHF) in patients with systemic lupus erythematosus (SLE) have received little attention. Recent data on the use of hydroxychloroquine in the treatment ...of SARS-CoV-2 infection have cast doubt on its cardiac safety. The factors associated with CHF, including therapy with antimalarials, were analyzed in a large multicenter SLE cohort.
Cross-sectional study including all patients with SLE (ACR-1997 criteria) included in the Spanish Society of Rheumatology Lupus Register (RELESSER), based on historically gathered data. Patients with CHF prior to diagnosis of SLE were excluded. A multivariable analysis exploring factors associated with CHF was conducted.
The study population comprised 117 patients with SLE (ACR-97 criteria) and CHF and 3,506 SLE controls. Ninety percent were women. Patients with CHF were older and presented greater SLE severity, organ damage, and mortality than those without CHF. The multivariable model revealed the factors associated with CHF to be ischemic heart disease (7.96 4.01–15.48, p < 0.0001), cardiac arrhythmia (7.38 4.00–13.42, p < 0.0001), pulmonary hypertension (3.71 1.84–7.25, p < 0.0002), valvulopathy (6.33 3.41–11.62, p < 0.0001), non-cardiovascular damage (1.29 1.16–1.44, p < 0.000) and calcium/vitamin D treatment (5.29 2.07–16.86, p = 0.0015). Female sex (0.46 0.25–0.88, p = 0.0147) and antimalarials (0.28 0.17–0.45, p < 0.000) proved to be protective factors.
Patients with SLE and CHF experience more severe SLE. Treatment with antimalarials appears to confer a cardioprotective effect.
Powdery mildew caused by Podosphaera xanthii has become a major problem in melon since it occurs all year round irrespective of the growing system. The TGR-1551 melon genotype was found to be ...resistant to several melon diseases, among them powdery mildew. However, the corresponding resistance genes have been never mapped. We constructed an integrated genetic linkage map using an F2 population derived from a cross between the multi-resistant genotype TGR-1551 and the susceptible Spanish cultivar ‘Bola de Oro'. The map spans 1,284.9 cM, with an average distance of 3.6 cM among markers, and consists of 354 loci (188 AFLP, 39 RAPD, 111 SSR, 14 SCAR/CAPS/dCAPS, and two phenotypic traits) distributed in 14 linkage groups. QTL analysis identified one major QTL (Pm-R) on LG V for resistance to races 1, 2, and 5 of powdery mildew. The PM4-CAPS marker is closely linked to the Pm-R QTL at a genetic distance of 1.9 cM, and the PM3-CAPS marker is located within the support interval of this QTL. These codominant markers, together with the map information reported here, could be used for melon breeding, and particularly for genotyping selection of resistance to powdery mildew in this vegetable crop species.
What is already known about this subject
• Flavonoids are largely recognized as potential inhibitors of platelet function, through nonspecific mechanisms such as antioxidant activity and/or ...inhibition of several enzymes and signalling proteins.
• In addition, we, and few others, have shown that certain antiaggregant flavonoids may behave as specific TXA2 receptor (TP) ligands in platelets.
• Whether flavonoids interact with TP isoforms in other cell types is not known, and direct evidence that flavonoid–TP interaction inhibits signalling downstream TP has not been shown.
What this study adds
• This study first demonstrates that certain flavonoids behave as ligands for both TP isoforms, not only in platelets, but also in human myometrium and in TP‐transfected HEK 293T cells.
• Differences in the effect of certain flavonoids in platelet signalling, induced by either U46619 or thrombin, suggest that abrogation of downstream TP signalling is related to their specific blockage of the TP, rather than to a nonspecific effect on tyrosine kinases or other signalling proteins.
Aims
Flavonoids may affect platelet function by several mechanisms, including antagonism of TxA2 receptors (TP). These TP are present in many tissues and modulate different signalling cascades. We explored whether flavonoids affect platelet TP signalling, and if they bind to TP expressed in other cell types.
Methods
Platelets were treated with flavonoids, or other selected inhibitors, and then stimulated with U46619. Similar assays were performed in aspirinized platelets activated with thrombin. Effects on calcium release were analysed by fluorometry and changes in whole protein tyrosine phosphorylation and activation of ERK 1/2 by Western blot analysis. The binding of flavonoids to TP in platelets, human myometrium and TPα‐ and TPβ‐transfected HEK 293T cells was explored using binding assays and the TP antagonist 3H‐SQ29548.
Results
Apigenin, genistein, luteolin and quercetin impaired U46619‐induced calcium mobilization in a concentration‐dependent manner (IC50 10–30 µm). These flavonoids caused a significant impairment of U46619‐induced platelet tyrosine phosphorylation and of ERK 1/2 activation. By contrast, in aspirin‐treated platelets all these flavonoids, except quercetin, displayed minor effects on thrombin‐induced calcium mobilization, ERK 1/2 and total tyrosine phosphorylation. Finally, apigenin, genistein and luteolin inhibited by >50% 3H‐SQ29548 binding to different cell types.
Conclusions
These data further suggest that flavonoids may inhibit platelet function by binding to TP and by subsequent abrogation of downstream signalling. Binding of these compounds to TP occurs in human myometrium and in TP‐transfected HEK 293T cells and suggests that antagonism of TP might mediate the effects of flavonoids in different tissues.
Summary
Platelet cold agglutinins (PCA) cause pseudothrombocytopenia, spurious thrombocytopenia due to
ex vivo
platelet clumping, complicating clinical diagnosis, but mechanisms and consequences of ...PCA are not well defined. Here, we characterised an atypical immunoglobulin (Ig)M PCA in a 37-year-old woman with lifelong bleeding and chronic moderate thrombocytopenia, that induces activation and aggregation of autologous or allogeneic platelets via interaction with platelet glycoprotein (GP)VI. Patient temperature-dependent pseudothrombocytopenia was EDTA-independent, but was prevented by integrin αIIbβ3 blockade. Unstimulated patient platelets revealed elevated levels of bound IgM, increased expression of activation markers (P-selectin and CD63), low GPVI levels and abnormally high thromboxane (TX)A
2
production. Patient serum induced temperature- and αIIbβ3-dependent decrease of platelet count in allogeneic donorcitrated platelet-rich plasma (PRP), but not in PRP from Glanzmann’s thrombasthenia or afibrinogenaemia patients. In allogeneic platelets, patient plasma induced shape change, P-selectin and CD63 expression,
14
C-serotonin release, and TXA
2
production. Activation was not inhibited by aspirin, cangrelor or blocking anti-Fc receptor (FcγRIIA) antibody, but was abrogated by inhibitors of Src and Syk, and by a soluble GPVI-Fc fusion protein. GPVI-deficient platelets were not activated by patient plasma. These data provide the first evidence for an IgM PCA causing platelet activation/aggregation via GPVI. The PCA activity persisted over a five-year follow-up period, supporting a causative role in patient chronic thrombocytopenia and bleeding.
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