Determinants of label non-adherence to non-vitamin K oral anticoagulants in patients with newly diagnosed atrial fibrillation.

Aims: To evaluate the extent and determinants of off-label non-vitamin K oral anticoagulant (NOAC) dosing in newly diagnosed Dutch AF patients. Methods and results: In the DUTCH-AF registry, patients with newly diagnosed AF (<6 months) are prospectively enrolled. Label adherence to NOAC dosing was assessed using the European Medicines Agency labelling. Factors associated with off-label dosing were explored by multivariable logistic regression analyses. From July 2018 to November 2020, 4500 patients were registered. The mean age was 69.6 ± 10.5 years, and 41.5% were female. Of the 3252 patients in which NOAC label adherence could be assessed, underdosing and overdosing were observed in 4.2% and 2.4%, respectively. In 2916 (89.7%) patients with a full-dose NOAC recommendation, 4.6% were underdosed, with a similar distribution between NOACs. Independent determinants (with 95% confidence interval) were higher age [odds ratio (OR): 1.01 per year, 1.01-1.02], lower renal function (OR: 0.96 per ml/min/1.73 m2, 0.92-0.98), lower weight (OR: 0.98 per kg, 0.97-1.00), active malignancy (OR: 2.46, 1.19-5.09), anaemia (OR: 1.73, 1.08-2.76), and concomitant use of antiplatelets (OR: 4.93, 2.57-9.46). In the 336 (10.3%) patients with a reduced dose NOAC recommendation, 22.9% were overdosed, most often with rivaroxaban. Independent determinants were lower age (OR: 0.92 per year, 0.88-0.96) and lower renal function (OR: 0.98 per ml/min/1.73 m2, 0.96-1.00). Conclusion: In newly diagnosed Dutch AF patients, off-label dosing of NOACs was seen in only 6.6% of patients, most often underdosing. In this study, determinants of off-label dosing were age, renal function, weight, anaemia, active malignancy, and concomitant use of antiplatelets.

Giant cell arteritis as a cardiovascular entity.

Giant cell arteritis (GCA) is a relatively infrequent disorder that is underdiagnosed and little appraised in the field of general cardiology. However, it is important to be familiar with the clinical picture of this disease, especially because of the risk of developing fatal aortic aneurysms. If the disease is suspected after a thorough history and clinical examination combined with laboratory investigation, the diagnosis can be confirmed with (18)F-2-deoxy-glucose positron emission tomographic (FDG-PET) imaging. Early recognition of giant cell arteritis followed by prompt treatment with glucocorticosteroids will decrease the risk of developing large-vessel complications. (Neth Heart J 2009;17:281-3.).

Acute coronary syndrome during pregnancy.

Two cases of myocardial infarction during the second trimester of pregnancy are presented. Both patients were treated with thrombolytic therapy. In the first case the treatment was successful, in the other patient thrombolysis only temporarily relieved the symptoms. Therefore, angiography was performed and a significant stenosis in the left anterior descending coronary artery was successfully treated with balloon angioplasty. Both women delivered a healthy child, the first child was born at term, the second at a gestational age of 33 weeks. Percutaneous intervention versus thrombolytic therapy in pregnancy is discussed.

The elongation domain of ELL is dispensable but its ELL-associated factor 1 interaction domain is essential for MLL-ELL-induced leukemogenesis.

The MLL-ELL chimeric gene is the product of the (11;19)(q23p13.1) translocation associated with de novo and therapy-related acute myeloid leukemias (AML). ELL is an RNA polymerase II elongation factor that interacts with the recently identified EAF1 (ELL associated factor 1) protein. EAF1 contains a limited region of homology with the transcriptional activation domains of three other genes fused to MLL in leukemias, AF4, LAF4, and AF5q31. Using an in vitro transformation assay of retrovirally transduced myeloid progenitors, we conducted a structure-function analysis of MLL-ELL. Whereas the elongation domain of ELL was dispensable, the EAF1 interaction domain of ELL was critical to the immortalizing properties of MLL-ELL in vitro. To confirm these results in vivo, we transplanted mice with bone marrow transduced with MLL fused to the minimal EAF1 interaction domain of ELL. These mice all developed AML, with a longer latency than mice transplanted with the wild-type MLL-ELL fusion. Based on these results, we generated a heterologous MLL-EAF1 fusion gene and analyzed its transforming potential. Strikingly, we found that MLL-EAF1 immortalized myeloid progenitors in the same manner as that of MLL-ELL. Furthermore, transplantation of bone marrow transduced with MLL-EAF1 induced AML with a shorter latency than mice transplanted with the MLL-ELL fusion. Taken together, these results indicate that the leukemic activity of MLL-ELL requires the EAF1 interaction domain of ELL, suggesting that the recruitment by MLL of a transactivation domain similar to that in EAF1 or the AF4/LAF4/AF5q31 family may be a critical common feature of multiple 11q23 translocations. In addition, these studies support a critical role for MLL partner genes and their protein-protein interactions in 11q23 leukemogenesis.

EAF1, a novel ELL-associated factor that is delocalized by expression of the MLL-ELL fusion protein.

The (11;19)(q23;p13.1) translocation in acute leukemia leads to the generation of a chimeric protein that fuses MLL to the transcriptional elongation factor ELL. A novel protein was isolated from a yeast 2-hybrid screen with ELL that was named EAF1 for ELL-associated factor 1. Using specific antibodies, the endogenous EAF1 and ELL proteins were coimmunoprecipitated from multiple cell lines. In addition, endogenous EAF1 also exhibited the capacity to interact with ELL2. Database comparisons with EAF1 identified a region with a high content of serine, aspartic acid, and glutamic acid residues that exhibited homology with the transcriptional activation domains of several translocation partner proteins of MLL, including AF4, LAF4, and AF5q31. A similar transcriptional activation domain has been identified in this region of EAF1. By confocal microscopy, endogenous EAF1 and ELL colocalized in a distinct nuclear speckled pattern. Transfection of the MLL-ELL fusion gene delocalized EAF1 from its nuclear speckled distribution to a diffuse nucleoplasmic pattern. In leukemic cell lines derived from mice transplanted with MLL-ELL-transduced bone marrow, EAF1 speckles were not detected. Taken together, these data suggest that expression of the MLL-ELL fusion protein may have a dominant effect on the normal protein-protein interactions of ELL.

Differential expression of the heavy-chain ferritin gene in non-adhered and adhered oligodendrocytes.

Oligodendrocytes (OLGs) synthesize and maintain central nervous system myelin. Little is known about the molecules and pathways involved in signaling the commencement of myelination; yet myelination is spatially and temporally controlled. We are interested in deciphering the signaling events that control the on and off switch of myelination. To address these questions we are using an in vitro model system consisting of pure cultures of OLGs, isolated from postmyelination brains. We have shown that in vitro, these OLGs regenerate and reenact the ontogenic development of myelin upon adhesion to a substratum; we have also shown that when transplanted in vivo, they can form myelin. Hence, identifying the genes that are turned on upon OLG adhesion seemed a worthwhile approach to singling out those genes that are critical to the differentiation process. For this we adopted a novel technique-differential display-that permits the comparison of gene expression under two sets of conditions; in our case between non-adhered and adhered OLGs. Using the differential display method in conjunction with a set of five arbitrary primers, we have identified five cDNAs that are activated upon OLG substratum adhesion. Four of these cDNAs proved to be fragments of the heavy (H)-chain ferritin cDNA. H-chain ferritin is involved in the rapid sequestration and delivery of iron; it is also a cytoprotectant. Brain iron is localized predominantly in OLGs. Our finding that the expression of H-chain ferritin is upregulated upon OLG regeneration/differentiation agrees with reports, in other cell types, that H-chain ferritin transcription is modulated by factors that control cell growth and differentiation. The enhanced transcription results in a twofold augmentation in ferritin synthesis. This is the first demonstration of an adherence-mediated activation of the H-chain ferritin gene. The observation has interesting implications. The transcription of the H-chain ferritin gene is associated with tissue stress such as might occur during rapid cell growth. The fact that this transcription takes place upon OLG-substratum adhesion can be viewed as another manifestation of the anchorage-induced signal that drives OLGs toward a differentiation program. This entails the synthesis of all the necessary components for the assembly of large quantities of membranes, hence constituting a period of rapid growth and considerable cell stress. We have sequenced the full-length ferritin cDNA of ovine OLGs; the cDNA is 775 bp long. The coding sequence starts at residue 57 with the ATG codon and terminates at residue 602, with the stop codon TAA. The cDNA codes for 181 amino acids with a predicted size for the protein of 22 kD. The deduced amino acid sequence is 93% identical to human ferritin. We postulate that OLG H-chain ferritin may function at more than one level: 1) it provides the iron required to sustain an oxidative metabolism; and 2) it acts as a cytoprotectant against oxidant-mediated injury.

GRASP: a novel heparin-binding serum glycoprotein that mediates oligodendrocyte-substratum adhesion.

Cell-substratum adhesion plays a crucial part in the cascade of events that control growth or turn on and consummate a differentiation program. We are investigating the molecular basis of oligodendrocyte (OLG) cytodifferentiation, employing pure cultures of OLGs isolated from postmyelination brains. We have shown that such OLGs will regenerate in vitro and reenact the ontogenic development of myelin, but to do so they need a signal. Adherence to a polylysine surface in the presence of 20% horse serum generates such a signal. Among the events that are turned on upon OLG adhesion is the phosphorylation of myelin basic protein; no such phosphorylation takes place in the non-adhered cell. We postulated that horse serum provides an adhesion molecule. Laminin, fibronectin, collagen and native vitronectin failed to replace horse serum. Hence, we set out to fractionate horse serum by screening with an adhesion assay. We report here the identification, purification and partial characterization of a novel, heparin-binding horse serum glycoprotein that we have termed Glycine-Rich Adhesion Serum Protein--GRASP--to stress the fact that this protein has a high content of glycine and functions, in vitro, as an adhesion molecule for OLGs. There is 61% similarity at the N-terminus between GRASP and histidine-rich glycoprotein precursor (HRGP), an alpha 2-glycoprotein from human plasma. However, our data suggest that GRASP is not the horse serum homolog of HRGP. First, the two Gps are functionally distinct: HRGP does not promote the adhesion of OLGs. Second, the amino acid compositions differ significantly, e.g., GRASP is not histidine- but rather glycine-rich. Third, the region of sequence similarity between GRASP and HRGP is conserved throughout the cystatin superfamily. Fourth, anti-Gp55 polyclonal Abs recognize a similar set of polypeptides--save for slight differences in M(r)-in human serum as in horse serum, indicating that HRGP and GRASP are two distinct but related proteins and are both present in human and horse sera. GRASP is a dimer trimer of seemingly identical subunits of M(r) approximately 55,000 ; the native protein has an M(r) x 10(-3) approximately 120-140, of which 24-27% is contributed by carbohydrate. Using GRASP as a substratum allows the growth of OLGs in serum-free medium. GRASP is as good an effector of myelin basic protein phosphorylation as 20% horse serum. We conjecture that the mechanism of GRASP function features: 1) exposure of a cryptic sequence--after a change in conformation induced upon binding to polylysine--with affinity for an OLG signal-transducing receptor; and 2) interaction of its heparin-binding domain with OLG surface heparin sulfate proteoglycans and/or the aforementioned receptor.

Myelin palingenesis. 1. Electron microscopical localization of myelin/oligodendrocyte proteins in multilamellar structures by the immunogold method.

The issue of the capacity of mature oligodendrocytes to remyelinate naked axons has not been totally resolved. The impression is that for this to happen, oligodendrocytes have to undergo cell division. We are interested in providing an answer to the question: can oligodendrocytes myelinate more than once? To address this question, we are using a model system consisting of pure cultures of postmyelination oligodendrocytes. We have previously shown that these cells have plasticity and are able to regenerate, but for this to occur they need a signal. In vitro, this signal is provided by interaction with a positively charged substratum and a serum factor(s). Over time in culture, without cell division, oligodendrocytes assemble multilamellar structures that ultrastructurally resemble myelin. We have named this process myelin palingenesis--rebirth--to distinguish it from remyelination. In order to ascertain the significance of the regenerative process observed in vitro and its predictive value as to the capabilities of oligodendrocytes to ensheathe axons, we undertook an ultrastructural and immunocytochemical characterization of the multilamellar membranes to assess whether they contain all the myelin-characteristic proteins, i.e., myelin basic protein, proteolipid protein, 2',3'-cyclic nucleotide 3'-phosphodiesterase and myelin-associated glycoprotein. We have used antibodies against these proteins as immunocytochemical probes in conjunction with the immunogold method at the electron microscopic level. Oligodendrocyte cultures were processed for electron microscopy. Blocks were serially sectioned parallel to the culture plate at 0.09-micron spacings. Staining of cells was done prior to embedding. Analysis of these micrographs brought to light the existence of membrane-membrane interaction between multilamellar structures; this is reminiscent of the interactions observed in situ between myelinated axons. We have found that the membranous structures possess all the myelin-characteristic proteins. Furthermore, based on the accessibility of these proteins to the antibodies, e.g., whether or not permeabilization was required, we can surmise that these proteins are incorporated into the membranous structures with the same orientation as in myelin. These results, in conjunction with our earlier work, show that mature oligodendrocytes stripped of their myelin are able to regenerate and reassemble multilamellar membranes that have all the myelin proteins. It remains to be proven that when provided with axons, these oligodendrocytes will generate typical myelin.

Indications for pacemaker implantation in the Kearns-Sayre syndrome.

The Kearns-Sayre syndrome is a rare condition, characterized by progressive external ophthalmoplegia, retinal pigmentary degeneration and progressive impairment of cardiac conduction, which mainly determines the prognosis. Two young patients (aged 13 and 18 years) without symptoms of cardiac disease presented with an electrocardiogram showing sinus rhythm, a normal atrio-ventricular conduction time, right bundle branch block and a left anterior fascicular block. Electrophysiologic investigation showed prolongation of His-ventricular interval at rest, which further increased during atrial pacing. Because of the potential progression of the conduction abnormalities and threatening sudden death, we decided to implant a pacemaker in both patients. Ten months later one patient had become pacemaker-dependent. Prophylactic pacemaker therapy is advisable in patients suffering from the Kearns-Sayre syndrome, who have bifascicular block on the precordial electrocardiogram.

Complete atrioventricular dissociation and His bundle tachycardia in a newborn: problems in prenatal diagnosis and postnatal management.

A newborn infant with complete atrioventricular dissociation and infranodal tachycardia, detected at 33 weeks gestation by fetal echocardiography, is described. In the perinatal period, infra or juxta-nodal tachycardia was noted, compromising the hemodynamic state of the newborn. A combination of flecainide and propranolol terminated the arrhythmia.

Plasma membrane of cultured oligodendrocytes: I. Isolation, purification, and initial characterization.

Oligodendrocytes generate myelin as extensions of the plasma membrane. Myelin has been well characterized, yet little is known concerning oligodendrocyte plasma membrane. We have developed a reproducible method for the isolation of an oligodendrocyte plasma membrane-rich fraction (F2.2). F2.2 has a 25-fold enrichment in K+-dependent p-nitrophenyl phosphatase, a plasma membrane marker. Impurities are composed of Golgi elements (8-12%), microsomes (4-6%), and lysosomal membranes (1-5%). Our starting material was oligodendrocytes kept in culture in a nonattached state for 3 to 5 d. After disrupting the cells and removing nuclei (P1), the supernatant (SP1) was fractionated on a self-generating 20% Percoll gradient into three bands: F1, F2, and F3. F1 had only 3% of the applied protein and was not characterized. F2, with 11% of the protein, was fivefold enriched in plasma membrane. F3 had 27% of initial protein; it consisted of a crude mitochondrial and lysosomal fraction. F2 was further purified by first washing it hypotonically, treating it with Mg2+, and then fractionating it on a Ficoll step gradient that yielded F2.2 at the interphase. Morphologically F2.2 comprises 1) membranous sheets, often with more than one membrane in close apposition; 2) membrane vesicles of various sizes and shapes frequently filled with amorphous material; 3) Golgi elements; and 4) unrecognizable profiles. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profile of F2.2 reveals CNPase as a major component in agreement with the high CNPase specific activity (3,860 mumol/mgP/h) found in F2.2. Other significant polypeptides have Mr = 170,000, 135,000, 108,000, 80,000, 53,000, 38,500, 32,000, and 22,200.

Plasma membrane of cultured oligodendrocytes: II. Possible structural and functional domains.

An oligodendrocyte plasma membrane-rich fraction, F2.2, was resolved by equilibrium density centrifugation on a linear sucrose gradient from 0.5 M to 1.3 M into three fractions, F2.2a, F.2.2b, F2.2c, and a pellet F2.2p. F2.2a and F.2.2b were enriched 1.5-fold relative to F2.2 in plasma membrane markers at the expense of F2.2c and F2.2p, which became correspondingly impoverished. This gave F2.2a and F2.2b a 42-fold and 37-fold enrichment, respectively, in plasma membrane markers relative to the initial cell homogenate. F2.2c had a sevenfold enrichment in a Golgi marker; together with F2.2p, they contained all the Golgi marker initially present in F2.2. Preliminary data indicated that the F2.2-subfractions differed from one another in their molar ratios of cholesterol to phospholipids and protein to lipids but had similar protein profiles when examined by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Their content of fucosylated glycoproteins appeared also to be different. Morphologically, F2.2a and F2.2b were very similar: they contained large membrane vesicles, membrane sheets, and vesicles entrapped within other vesicles. Membrane-membrane interaction was apparent in these fractions. F2.2c had many of the same elements, but most of the membrane structures contained amorphous material. F2.2p differed morphologically from the other fractions in that it had principally electron-dense structures. It is postulated that F2.2a, F2.2b, and perhaps F2.2c represent different domains of oligodendrocyte plasma membrane. Alternatively, these fractions might correspond to the plasma membrane of oligodendrocyte subtypes.

Plasma membrane of cultured oligodendrocytes: III. Relatedness to myelin.

We have compared highly purified fractions of oligodendrocyte plasma membrane to myelin by one- and two dimensional gel electrophoresis and found them to be distinct. The major myelin proteins--proteolipid protein (PLP), DM-20, and myelin basic protein (MBP), which dominate the sodium dodecyl sulfate polyacrylamide gel electrophoresis pattern of myelin--were minor components of the plasmalemma. However, 2',3', cyclic nucleotide phosphodiesterase (CNPase) and myelin-associated glycoprotein (MAG) were represented equally in both membranes. Labeling the cells with various precursors followed by isolation of plasmalemma revealed that newly synthesized PLP, DM-20, CNPase, and MAG were incorporated into the plasma membrane of "floating" oligodendrocytes (i.e., nonattached to substratum). This was not so with MBP. Nevertheless, scattered patches of MBP were localized on the plasma membrane of intact cells using the immunogold method at the electron microscopic level. The data are consistent with the notion that MBP is not a constituent of the plasma membrane of mature oligodendrocytes (the MBP patches on intact cells are likely remnants from past association with myelin) but is rapidly associated with the plasmalemma of myelinating oligodendrocytes (i.e., attached cells). It is suggested that phosphorylation of MBP provides the triggering signal for plasma membrane association. In order to analyze the minor proteins in myelin and compare them to the plasma membrane by two-dimensional gel electrophoresis, myelin was extracted with chloroform:methanol to remove PLP, DM-20, and MBP. Even in the absence of PLP, DM-20, and MBP the pattern of extracted myelin still differed from that of plasmalemma indicating that their minor protein compositions were not the same. Myelin was characterized by a group of proteins that clustered at pI 5.5-6.5 and Mr 40,000-60,000 of which alpha-tubulins, beta-tubulins, and actin are part: the plasmalemma had tubulins and actin but in different proportions. Our findings indicate that in addition to PLP, DM-20, and MPB, myelin is also enriched relative to the plasmalemma in another group of proteins.

Transoesophageal cross-sectional echocardiographic recognition of an aortic valve ring abscess and a subannular mycotic aneurysm.

We present the apparently unique transoesophageal cross-sectional echocardiographic features of a mycotic aneurysm of the left ventricular outflow tract communicating with an abscess in the anterior free wall of the left ventricle. Precordial echocardiographic studies had been hampered by the interposition of an aortic valve prosthesis.

Cultured oligodendrocytes. A role for cell-substratum interaction in phenotypic expression.

Oligodendrocytes can be maintained in two states: nonattached; we call these cells B3.f; morphologically they resemble freshly isolated cells; attached; we refer to the latter as B3.fA. Profound morphological, ultrastructural, and biochemical changes take place upon adhesion to a competent surface (Szuchet, S., Yim, S. H., and Monsma, S. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 7019-7023). Here we present evidence that the transition from B3.f to B3.fA has important consequences for the expression of myelinogenic properties by these cells. We have examined the incorporation of [3H]leucine, [35S]methionine, and [35S]cysteine into polypeptide chains by B3.f and B3.fA cells from 3 days after isolation up to 8 weeks in culture. Specific antisera against myelin and cytoskeletal proteins were used to identify the newly synthesized proteins. Our results indicate that: overall incorporation expressed as cpm/mg of protein remains essentially constant and independent of the state of adhesion or time in culture; B3.f cells keep a low profile in the synthesis of the major myelin proteins but have a high uptake of precursors into 2',3'-cyclic nucleotide phosphodiesterase, actin, and tubulin; adhesion of oligodendrocytes to a polylysine substratum activates the synthesis and phosphorylation of myelin basic protein, and the synthesis and acylation of proteolipid protein and DM-20; over time in culture there is an increased synthesis and accumulation of these proteins and of myelin-associated glycoprotein. We conclude that B3.f cells exhibit a behavior that is distinct from that of B3.fA cells. Our results are consistent with the notion that upon adhesion to a substratum, oligodendrocytes undergo a transition from myelin-maintaining cells (B3.f) to that of myelin-forming cells (B3.fA). This conclusion is substantiated by the finding of myelin membranes in these cultures.

Mature oligodendrocytes cultured in the absence of neurons recapitulate the ontogenic development of myelin membranes.

We have maintained isolated oligodendrocytes in culture for up to 5 months. Over time cultured cells acquire conspicuous membranous profiles that either emanate from cell processes and perikarya or are apposed to them. These membranous profiles have biochemical and ultrastructural features characteristic of myelin. Because the cells used in this study are post-myelination (cells are isolated from white matter of mature brains), we argue that this is a case of resynthesis of myelin membranes. Because this process takes place in the absence of neurons, we have termed it myelin palingenesis in order to distinguish it from remyelination, a term commonly used to designate reensheathment of axons. Myelin palingenesis may occur seemingly without wrapping and spiraling, but membranes wrapped around oligodendrocyte processes and soma are also observed. Irrespective of the modes of formation, those membranes that are compact have the fine structure of normal myelin. Thus, long-term cultures of mature oligodendrocytes should be a good model system in helping to decipher the molecular and cellular mechanisms involved in myelination and in remyelination.

A simple cell disrupter designed for small cells with relatively large nuclei.

A simple cell disrupter that is particularly suitable for breaking small cells with relatively large nuclei is described. Cells are disrupted by the shearing forces set up as they are pushed by a positive N2 pressure through a 0.8- to 1.5-micrometer stainless-steel filter. The total procedure takes only a few minutes, it is highly reproducible, and the yield is good. The cell homogenate obtained is a good starting source for the isolation of plasma membranes, intracellular organelles, and nuclei.