In the pathogenesis of bacterial endocarditis (BE), the clotting system plays a cardinal role in ... more In the pathogenesis of bacterial endocarditis (BE), the clotting system plays a cardinal role in the formation and maintenance of the endocardial vegetations. The extrinsic pathway is involved in the activation of the coagulation pathway with tissue factor (TF) as the key protein. Staphylococcus aureus is a frequently isolated bacterium from patients with BE. We therefore investigated whether S. aureus can induce TF activity (TFA) on fibrin-adherent monocytes, used as an in vitro model of BE. We also assessed in vivo in rabbits with catheter induced vegetations, the effect of S. aureus infection on vegetational TFA. In vitro experiments showed that adherent S. aureus induced TFA on fibrin-adherent monocytes which was optimal at a bacterium/monocyte ratio of 1 to 1. Monocyte damage occurred when this ratio exceeded 4 to 1 (visually) or 6 to 1 (propidium iodide influx) Consequently, TFA decreased. In vivo S. aureus led to very high bacterial numbers in the vegetations and a significan...
A main feature in the pathogenesis of bacterial endocarditis is the activation of the coagulation... more A main feature in the pathogenesis of bacterial endocarditis is the activation of the coagulation system via the extrinsic pathway, resulting in the formation of infected endocardial vegetations. Earlier studies gave indirect evidence that monocytes play an important role in the procoagulant response during the course of the disease. In this study, we assessed the role of monocytes more directly. We compared weights and tissue factor activities (TFA) of endocardial vegetations of normal rabbits infected with Streptococcus sanguis with those of rabbits which were treated with the cytostatic drug etoposide (Vepesid; Bristol-Myers Squibb B.V.) to induce a selective monocytopenia. Furthermore, the importance of the presence of bacteria was determined through the influence of antibiotic treatment on TFA, vegetational weight, and infection of the vegetations. The TFA of the vegetations was measured chromogenically by monitoring the factor VII-dependent activation of factor X with an amido...
The endocardial vegetation which is formed in the course of bacterial endocarditis (BE) contains ... more The endocardial vegetation which is formed in the course of bacterial endocarditis (BE) contains tissue factor (TF)-dependent procoagulant activity. Earlier studies showed that monocytes are the main source of TF in the vegetations. The TF activity (TFA) of vegetations isolated from Streptococcus sanguis-infected rabbits depended on the numbers of bacteria as well as monocytes in the vegetation. In this study, we investigated whether forStaphylococcus epidermidis, a frequent pathogen in BE, an effect similar to that found for S. sanguis could be shown. In vitro, S. epidermidis was found to stimulate TFA of fibrin adherent monocytes significantly. This stimulation was maximal at a bacterium-to-monocyte ratio of 7. In vivo, TFA was found to be significantly higher in S. epidermidis-infected than in sterile catheter-induced vegetations. Reduction of vegetational bacterial numbers by teicoplanin treatment lead to a small but significant decrease of TFA. Reduction of monocyte numbers by ...
A cardinal process in bacterial endocarditis (BE) is the activation of the clotting system and th... more A cardinal process in bacterial endocarditis (BE) is the activation of the clotting system and the formation of a fibrin clot on the inner surface of the heart, the so-called endocardial vegetation. The processes that lead to the activation of the clotting system on endothelial surfaces upon exposure to bacteria are largely unknown. In the present study, we investigated in an in vitro model whether infection of human endothelial cells (EC) with bacteria that are relevant to BE, such as Staphylococcus aureus,Streptococcus sanguis, and Staphylococcus epidermidis, leads to induction of tissue factor (TF)-dependent procoagulant activity (TFA) and whether this process is influenced by host factors, such as interleukin-1 (IL-1), that are produced in response to the bacteremia in vivo. The results show that S. aureus binds to and is internalized by EC, resulting in expression of TF mRNA and TF surface protein as well as generation of TFA within 4 to 8 h after infection. No TFA was found wh...
Intravascular infection with Staphylococcus aureus, Staphylococcus epidermidis, or Streptococcus ... more Intravascular infection with Staphylococcus aureus, Staphylococcus epidermidis, or Streptococcus sanguis can initiate fibrin formation on endocardial tissue, causing bacterial endocarditis. The ability of these bacteria to injure intact endothelial cells (ECs) and to aggravate tissue factor (TF)-dependent coagulation in the presence of blood leukocytes was investigated. Cytolysis of ECs occurred after infection with S. aureus and, with membrane-bound monocytes or granulocytes present, also after infection with S. sanguis or S. epidermidis. Monocytes that subsequently bound to the resultant bacteria-infected subcellular EC matrix (ECM) elicited TF mRNA, TF antigen, and TF activity (TFA). This was most pronounced in ECM prepared after the cytolysis of ECs by infection with S. aureus or S. epidermidis. We demonstrate that monocytes continue and intensify fibrin formation after lysis of bacteria-infected ECs, which suggests that, during the course of intravascular infection, early fibrin formation shifts from being mediated by EC-derived TFA to being mediated by TFA of monocytes bound to bacteria-infected ECM.
In bacterial endocarditis (BE), intravascular infection with Staphylococcus aureus , Streptococcu... more In bacterial endocarditis (BE), intravascular infection with Staphylococcus aureus , Streptococcus sanguis, or Staphylococcus epidermidis can lead to formation of a fibrin clot on the inner surface of the heart and cause heart dysfunction. The events that start the coagulation in the early stage of the disease are largely unknown. We have recently shown that human endothelial cells (EC) upon binding and internalization of S. aureus , but not S. sanguis or S. epidermidis , express tissue factor (TF)-dependent procoagulant activity (TFA). The present study shows that infection of EC with these three pathogens induces surface expression of intracellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) and monocyte adhesion. Subsequent coculture of these cells synergistically enhanced TFA, which was exclusively dependent on TF molecules that were expressed on EC during coculture. TFA induction required direct contact between monocytes and bacterium-infected EC...
Staphylococcus aureus is isolated from a substantial number of patients with infective endocardit... more Staphylococcus aureus is isolated from a substantial number of patients with infective endocarditis who are not known to have predisposing heart abnormalities. It has been suggested that the infection is initiated by the direct binding of S. aureus to human vascular endothelium. To determine the mutual response of the endothelial cells and the bacteria, we studied the interaction between S. aureus and human vascular endothelium. Scanning electron microscopic analyses showed that binding of S. aureus to human umbilical vein endothelial cells (HUVEC) mainly occurred via thread-like protrusions extending from the cell surface. Bound bacteria appeared to be internalized via retraction of the protrusions into newly formed invaginations of the endothelial cell surface. The growth phase of S. aureus had a major impact on the interaction with HUVEC. Logarithmically growing bacteria showed increased binding to, and were more readily internalized by, HUVEC compared to stationary-phase bacteri...
In the pathogenesis of bacterial endocarditis (BE), the clotting system plays a cardinal role in ... more In the pathogenesis of bacterial endocarditis (BE), the clotting system plays a cardinal role in the formation and maintenance of the endocardial vegetations. The extrinsic pathway is involved in the activation of the coagulation pathway with tissue factor (TF) as the key protein. Staphylococcus aureus is a frequently isolated bacterium from patients with BE. We therefore investigated whether S. aureus can induce TF activity (TFA) on fibrin-adherent monocytes, used as an in vitro model of BE. We also assessed in vivo in rabbits with catheter induced vegetations, the effect of S. aureus infection on vegetational TFA. In vitro experiments showed that adherent S. aureus induced TFA on fibrin-adherent monocytes which was optimal at a bacterium/monocyte ratio of 1 to 1. Monocyte damage occurred when this ratio exceeded 4 to 1 (visually) or 6 to 1 (propidium iodide influx) Consequently, TFA decreased. In vivo S. aureus led to very high bacterial numbers in the vegetations and a significan...
Monocytes are important effector cells in the pathogenesis of bacterial endocarditis since they p... more Monocytes are important effector cells in the pathogenesis of bacterial endocarditis since they provide the tissue factor that activates the coagulation system and maintains established vegetations. Monocytes secrete cytokines that can modulate monocyte tissue factor activity (TFA), thereby affecting the formation and maintenance of vegetations. In this study, we show that monocytes cultured for 4 h on a Streptococcus sanguis -infected fibrin matrix mimicking the in vivo vegetational surface express high levels of TFA. This was accompanied by secretion of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-1α (IL-1α), and IL-1β. After a 24-h incubation period the anti-inflammatory cytokine IL-10 could also be detected. Our data show that, whereas TNF-α and IL-1 have a minor role in the induction of TFA by monocytes cultured on a fibrin matrix, TNF-α but not IL-1 plays an important role in the induction of IL-10 by these cells. In turn, our data show that I...
CD44 variants but when the cells were activated with PMA, expression of exons v3andvBwasdetected.... more CD44 variants but when the cells were activated with PMA, expression of exons v3andvBwasdetected. Conclusions: These data indicate expression of multiple forms of CD44 constitutively on PBMC and suggest that they may have a role to play in normal T-ceil function. However expressfon of v3 and VB was found to be upregulated on Flow Cytometric analysts following PMA stimulation suggesting that, on cellular activation, there may be a conformational change with exposure of epitopes mcognised by these antibodies. Conduslon: TGFB promotes the apoptosis of post-activated T cells and may be a regulatory mechanism acting on extravasated lymphocytes in viva. TGFB may have a role in the microenvimnmental regulation of migratory cells, as it promotes the post-activation induced apoptosis of T cells when added at peak T cell activation. Our results show that TGF/l had no direct effect on T cell activation per se, thereby confirming previous repotts. The potent immunosuppressive and anti-inflammatory effects ascribed to TGFfi may be partly due to the promotion of apoptosis, rather than any direct suppressive effect.
<p>Pups were analysed for chimerism by multiplex PCR and MCA. <b>A</b>) Four of... more <p>Pups were analysed for chimerism by multiplex PCR and MCA. <b>A</b>) Four of the pups derived after transplantation of blastocysts injected with ES cells of clone 9B4 showed presence of the exon 52 deleted <i>hDMD</i> gene (lines 1, 4, 8 and 11). <b>B</b>) Melting curve analysis revealed that all male pups were also chimeric for the <i>mdx</i> point mutation.</p
<p><b>A</b>) Nested PCR revealing exon skipping upon local 51ViM or 53ViM treat... more <p><b>A</b>) Nested PCR revealing exon skipping upon local 51ViM or 53ViM treatment in right and left gastrocnemius and triceps muscle (respectively GR, GL and TR, TL) of two del52hDMD<i>/mdx</i>#35 mice. We confirmed by Sanger sequencing that the upper band in the untreated del52hDMD<i>/mdx</i> samples involves a cryptic splicing event that is occasionally observed in untreated mice of this strain. It contains exon 51, part of intron 51, the last 101 nucleotides of exon 52, exon 53 and multiple stop codons. The arrows indicate the expected heights of fragments lacking exon 51 (700 bp) or exon 53 (721 bp). <b>B</b>) Murine specific nested PCR confirmed that the exon 51 ViM induced low levels of mouse exon 51 skipping. The exon 53 ViM only resulted in exon 53 skipping in the human transcript as no skipping band was seen in the PCR performed with mouse-specific primers (expected size 483 bp). Sanger sequence confirmed that the smaller fragment obtained in the ViM exon 51 treated muscles contained the boundary of exon 50–52. Human ctrl; healthy human control sample. The arrow indicates the expected height of fragments lacking exon 51 (463 bp). <b>C-D</b>) Exon skipping resulted in the restored dystrophin expression in gastrocnemius (C) and triceps (D) of 51ViM and 53ViM treated mice.</p
<p><b>A</b>) Western blot analyses of heart and quadriceps, incubated with eith... more <p><b>A</b>) Western blot analyses of heart and quadriceps, incubated with either GTX (human and mouse specific) or Mandys106 (human specific). Wild type expression levels of human dystrophin were observed in hDMD/<i>mdx</i> mice. Notably, del52hDMD/<i>mdx</i>#37 mice expressed traces of human dystrophin, in both cardiac and skeletal muscle, while this was not observed in del52hDMD/<i>mdx</i>#35 and <i>mdx</i>(BL6) mice. <b>B</b>) Sections of the heart and quadriceps stained with human specific dystrophin antibodies. Expression of human dystrophin is at wild type level in hDMD/<i>mdx</i> mice as anticipated. Both C57BL/6J, <i>mdx</i>(BL6) and del52hDMD/<i>mdx</i>#35 mice did not express human dystrophin. Interestingly, in most fibers of del52hDMD/<i>mdx</i>#37 mice, human dystrophin was expressed at low levels. Haematoxylin and eosin staining revealed signs of degeneration and regeneration in the quadriceps of both del52hDMD/<i>mdx</i> strains, as evident by variation in fiber size, centralized nuclei and patches of fibrosis and inflammation. Overall pathology appeared to be slightly less extensive in del52hDMD/<i>mdx</i>#37 mice compared to <i>mdx</i>(BL6) and del52hDMD/<i>mdx</i>#35 mice. <b>C</b>) Almost no centralized nuclei were found in wild type mice, while half of the myofibers in <i>mdx</i>(BL6) and del52hDMD/<i>mdx</i>#35 mice had centrally located nuclei. The percentage in del52hDMD/<i>mdx</i>#37 mice was with 26% significantly lower. Data were based on manual counts of 5 randomly taken pictures of 2 males and 2 females per genotype. Asterisks indicate <i>P</i><0.01.</p
<p><b>A</b>) Forelimb grip strength was impaired in <i>mdx</i>(BL6)... more <p><b>A</b>) Forelimb grip strength was impaired in <i>mdx</i>(BL6) and del52hDMD<i>/mdx</i> mice. <b>B</b>) hDMD/<i>mdx</i> mice were resistant against fatigue, while muscles of <i>mdx</i>(BL6) and del52hDMD<i>/mdx</i> mice were fatigued at the end of the grip strength protocol. Performance in hanging tests starting with two <b>C</b>) and four limbs <b>D</b>) was impaired in <i>mdx</i>(BL6) and del52hDMD<i>/mdx</i> mice. Overall, del52hDMD<i>/mdx</i>#37 mice outperformed <i>mdx</i>(BL6) and del52hDMD<i>/mdx</i>#35 mice. <b>E</b>) Creatine kinase levels were elevated in both del52hDMD<i>/mdx</i> and <i>mdx</i>(BL6) strains compared to hDMD/<i>mdx</i> mice. Asterisk indicates <i>P</i><0.05, data are represented as the mean ± SEM. hDMD/<i>mdx</i>, <i>mdx</i>(BL6) and del52hDMD<i>/mdx</i>#35 groups consisted of n = 3 males and n = 2 females (one male <i>mdx</i>(BL6) mouse died), while the del52hDMD<i>/mdx</i>#37 group consisted of n = 2 males and n = 3 females.</p
<p>Single ES clones were cultured in 96-well plates and DNA was isolated and used as templa... more <p>Single ES clones were cultured in 96-well plates and DNA was isolated and used as template in a multiplex PCR. Here the exons 46, 51 and 52 of the <i>hDMD</i> gene were analysed where exon 46 and 51 are positive controls and exon 52 the target to be deleted. <b>A</b>) An example is shown where candidate samples 2 and 5 are of interest because they are negative for exon 52 but positive for the control exons. <b>B</b>) For a large number of clones additional fragments were found for exon 52, suggesting non-homologues end joining (NHEJ) of TALEN induced double stranded breaks <b>C</b>) Representative image of LR-PCR performed on DNA of sub-clones of four exon 52 negative clones (9B4, 10H2, 11C9 and 11E7). LR-PCR was performed with primers targeting intron 51 (outside the targeting arm) and blasticidin (only present after homologous recombination), to rule out loss of PCR primer recognition sites by NHEJ and to confirm true targeting. <b>D</b>) RT-PCR was performed for RNA isolated from embryoid bodies of selected clones. The different fragments were isolated, purified and Sanger sequence analysed. In the wild type situation exon 52 was present, whereas in the properly targeted clones exon 52 was not present. This confirmed the exon 52 deletion on RNA level.</p
Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease generally caused by reading ... more Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease generally caused by reading frame disrupting mutations in the DMD gene resulting in loss of functional dystrophin protein. The reading frame can be restored by antisense oligonucleotide (AON)-mediated exon skipping, allowing production of internally deleted, but partially functional dystrophin proteins as found in the less severe Becker muscular dystrophy. Due to genetic variation between species, mouse models with mutations in the murine genes are of limited use to test and further optimize human specific AONs in vivo. To address this we have generated the del52hDMD/mdx mouse. This model carries both murine and human DMD genes. However, mouse dystrophin expression is abolished due to a stop mutation in exon 23, while the expression of human dystrophin is abolished due to a deletion of exon 52. The del52hDMD/mdx model, like mdx, shows signs of muscle dystrophy on a histological level and phenotypically mild functional impairment. Local administration of human specific vivo morpholinos induces exon skipping and dystrophin restoration in these mice. Depending on the number of mismatches, occasional skipping of the murine Dmd gene, albeit at low levels, could be observed. Unlike previous models, the del52hDMD/mdx model enables the in vivo analysis of human specific AONs targeting exon 51 or exon 53 on RNA and protein level and muscle quality and function. Therefore, it will be a valuable tool for optimizing human specific AONs and genome editing approaches for DMD.
Duchenne muscular dystrophy (DMD) is a muscle-wasting disease in which muscle is continuously dam... more Duchenne muscular dystrophy (DMD) is a muscle-wasting disease in which muscle is continuously damaged, resulting in loss of muscle tissue and function. Antisense-mediated exon skipping is a promising therapeutic approach for DMD. This method uses sequence specific antisense oligonucleotides (AONs) to reframe disrupted dystrophin transcripts. As AONs function in a sequence specific manner, human specific AONs cannot be tested in the mdx mouse, which carries a mutation in the murine Dmd gene. We have previously generated a mouse model carrying the complete human DMD gene (hDMD mouse) integrated in the mouse genome to overcome this problem. However, as this is not a disease model, it cannot be used to study the effect of AON treatment on protein level and muscle function. Therefore, our long term goal is to generate deletions in the human DMD gene in a mouse carrying the hDMD gene in an mdx background. Towards this aim, we generated a male ES cell line carrying the hDMD gene while having the mdx point mutation. Inheritance of the hDMD gene by the ES cell was confirmed both on DNA and mRNA level. Quality control of the ES cells revealed that the pluripotency marker genes Oct-4 and Nanog are well expressed and that 85% of cells have 40 chromosomes. Germ line competence of this cell line has been confirmed, and 2 mice strains were derived from this cell line and crossed back on a C57BL6 background: hDMD/mdx and mdx(BL6). Funding Statement MV and LV are paid by a grant from the Prinses Beatrix Spierfonds (the Netherlands) and AAR receives funding from ZonMw, the Prinses Beatrix Spierfonds and the Duchenne Parent Project (the Netherlands). The infrastructure of the Center for Medical Systems Biology and the Center for Biomedical Genetics was utilized to conduct the reported work. JK, JC, AAR, and JV report being employed by LUMC, which has patents on exon skipping. As a co-author on some of these patents AAR is entitled to a share of royalties. MV, JK, JC, LV and JV declare no conflict of interest. Research groups are exploring various therapies to slow down disease progression. Therapies are mainly based on restoration of dystrophin expression and on improving muscle quality. Antisense-mediated exon skipping, aiming to convert a severe DMD phenotype into a milder Becker muscular dystrophy phenotype is currently closest to clinical application 5. In DMD patients the reading-frame of the dystrophin mRNA is disrupted resulting in a prematurely truncated, non functional dystrophin protein. Utilizing antisense oligonucleotides (AONs), the splicing of an out of frame exon during maturation of the pre-mRNA is prevented to restore the reading frame, allowing the production of an internally deleted but partially functional dystrophin protein. AONs target exons in a sequence specific manner and therefore human specific AONs cannot be tested in mouse models 6. Consequently, the mdx mouse, which carries a mutation in exon 23 of the murine Dmd gene and is the most widely used model for DMD research, cannot be used as a preclinical model to test human specific AONs. Therefore we have developed the hDMD mouse 7 , which carries the complete human DMD (hDMD) gene integrated in the mouse genome (B6.DBA2.129-hDMD tg/tg , from now on referred to as hDMD mouse). When crossed with mdx mice, the human dystrophin can compensate for the lack of mouse dystrophin and prevent pathology 7. A drawback of the hDMD mouse is that it is not a disease model, due to its expression of human dystrophin. This makes it impossible to study the restoration of human dystrophin and muscle function and quality upon AON treatment. A mouse model to allow in vivo assessment of human specific AONs would be very valuable. Preferably this model would not express mouse dystrophin and would have a deletion in the human DMD gene (?hDMD) in the mutation hotspot region (between exon 45 and
Rare diseases can be caused by genetic mutations that disrupt normal pre-mRNA splicing. Antisense... more Rare diseases can be caused by genetic mutations that disrupt normal pre-mRNA splicing. Antisense oligonucleotide treatment to the splicing thus has therapeutic potential for many rare diseases. In this review we will focus on the state of the art on exon skipping using antisense oligonucleotides as a potential therapy for rare genetic diseases, outlining how this versatile approach can be exploited to correct for different mutations.
Motivation: Advances in sequencing technologies and computational algorithms have enabled the stu... more Motivation: Advances in sequencing technologies and computational algorithms have enabled the study of genomic variants to dissect their functional consequence. Despite this unprecedented progress, current tools fail to reliably detect and characterize more complex allelic variants, such as short tandem repeats (STRs). We developed TSSV as an efficient and sensitive tool to specifically profile all allelic variants present in targeted loci. Based on its design, requiring only two short flanking sequences, TSSV can work without the use of a complete reference sequence to reliably profile highly polymorphic, repetitive or uncharacterized regions. Results: We show that TSSV can accurately determine allelic STR structures in mixtures with 10% representation of minor alleles or complex mixtures in which a single STR allele is shared. Furthermore, we show the universal utility of TSSV in two other independent studies: characterizing de novo mutations introduced by transcription activator-like effector nucleases (TALENs) and profiling the noise and systematic errors in an IonTorrent sequencing experiment. TSSV complements the existing tools by aiding the study of highly polymorphic and complex regions and provides a high-resolution map that can be used in a wide range of applications, from personal genomics to forensic analysis and clinical diagnostics. Availability and implementation: We have implemented TSSV as a Python package that can be installed through the command-line using pip install TSSV command. Its source code and documentation are
In the pathogenesis of bacterial endocarditis (BE), the clotting system plays a cardinal role in ... more In the pathogenesis of bacterial endocarditis (BE), the clotting system plays a cardinal role in the formation and maintenance of the endocardial vegetations. The extrinsic pathway is involved in the activation of the coagulation pathway with tissue factor (TF) as the key protein. Staphylococcus aureus is a frequently isolated bacterium from patients with BE. We therefore investigated whether S. aureus can induce TF activity (TFA) on fibrin-adherent monocytes, used as an in vitro model of BE. We also assessed in vivo in rabbits with catheter induced vegetations, the effect of S. aureus infection on vegetational TFA. In vitro experiments showed that adherent S. aureus induced TFA on fibrin-adherent monocytes which was optimal at a bacterium/monocyte ratio of 1 to 1. Monocyte damage occurred when this ratio exceeded 4 to 1 (visually) or 6 to 1 (propidium iodide influx) Consequently, TFA decreased. In vivo S. aureus led to very high bacterial numbers in the vegetations and a significan...
A main feature in the pathogenesis of bacterial endocarditis is the activation of the coagulation... more A main feature in the pathogenesis of bacterial endocarditis is the activation of the coagulation system via the extrinsic pathway, resulting in the formation of infected endocardial vegetations. Earlier studies gave indirect evidence that monocytes play an important role in the procoagulant response during the course of the disease. In this study, we assessed the role of monocytes more directly. We compared weights and tissue factor activities (TFA) of endocardial vegetations of normal rabbits infected with Streptococcus sanguis with those of rabbits which were treated with the cytostatic drug etoposide (Vepesid; Bristol-Myers Squibb B.V.) to induce a selective monocytopenia. Furthermore, the importance of the presence of bacteria was determined through the influence of antibiotic treatment on TFA, vegetational weight, and infection of the vegetations. The TFA of the vegetations was measured chromogenically by monitoring the factor VII-dependent activation of factor X with an amido...
The endocardial vegetation which is formed in the course of bacterial endocarditis (BE) contains ... more The endocardial vegetation which is formed in the course of bacterial endocarditis (BE) contains tissue factor (TF)-dependent procoagulant activity. Earlier studies showed that monocytes are the main source of TF in the vegetations. The TF activity (TFA) of vegetations isolated from Streptococcus sanguis-infected rabbits depended on the numbers of bacteria as well as monocytes in the vegetation. In this study, we investigated whether forStaphylococcus epidermidis, a frequent pathogen in BE, an effect similar to that found for S. sanguis could be shown. In vitro, S. epidermidis was found to stimulate TFA of fibrin adherent monocytes significantly. This stimulation was maximal at a bacterium-to-monocyte ratio of 7. In vivo, TFA was found to be significantly higher in S. epidermidis-infected than in sterile catheter-induced vegetations. Reduction of vegetational bacterial numbers by teicoplanin treatment lead to a small but significant decrease of TFA. Reduction of monocyte numbers by ...
A cardinal process in bacterial endocarditis (BE) is the activation of the clotting system and th... more A cardinal process in bacterial endocarditis (BE) is the activation of the clotting system and the formation of a fibrin clot on the inner surface of the heart, the so-called endocardial vegetation. The processes that lead to the activation of the clotting system on endothelial surfaces upon exposure to bacteria are largely unknown. In the present study, we investigated in an in vitro model whether infection of human endothelial cells (EC) with bacteria that are relevant to BE, such as Staphylococcus aureus,Streptococcus sanguis, and Staphylococcus epidermidis, leads to induction of tissue factor (TF)-dependent procoagulant activity (TFA) and whether this process is influenced by host factors, such as interleukin-1 (IL-1), that are produced in response to the bacteremia in vivo. The results show that S. aureus binds to and is internalized by EC, resulting in expression of TF mRNA and TF surface protein as well as generation of TFA within 4 to 8 h after infection. No TFA was found wh...
Intravascular infection with Staphylococcus aureus, Staphylococcus epidermidis, or Streptococcus ... more Intravascular infection with Staphylococcus aureus, Staphylococcus epidermidis, or Streptococcus sanguis can initiate fibrin formation on endocardial tissue, causing bacterial endocarditis. The ability of these bacteria to injure intact endothelial cells (ECs) and to aggravate tissue factor (TF)-dependent coagulation in the presence of blood leukocytes was investigated. Cytolysis of ECs occurred after infection with S. aureus and, with membrane-bound monocytes or granulocytes present, also after infection with S. sanguis or S. epidermidis. Monocytes that subsequently bound to the resultant bacteria-infected subcellular EC matrix (ECM) elicited TF mRNA, TF antigen, and TF activity (TFA). This was most pronounced in ECM prepared after the cytolysis of ECs by infection with S. aureus or S. epidermidis. We demonstrate that monocytes continue and intensify fibrin formation after lysis of bacteria-infected ECs, which suggests that, during the course of intravascular infection, early fibrin formation shifts from being mediated by EC-derived TFA to being mediated by TFA of monocytes bound to bacteria-infected ECM.
In bacterial endocarditis (BE), intravascular infection with Staphylococcus aureus , Streptococcu... more In bacterial endocarditis (BE), intravascular infection with Staphylococcus aureus , Streptococcus sanguis, or Staphylococcus epidermidis can lead to formation of a fibrin clot on the inner surface of the heart and cause heart dysfunction. The events that start the coagulation in the early stage of the disease are largely unknown. We have recently shown that human endothelial cells (EC) upon binding and internalization of S. aureus , but not S. sanguis or S. epidermidis , express tissue factor (TF)-dependent procoagulant activity (TFA). The present study shows that infection of EC with these three pathogens induces surface expression of intracellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) and monocyte adhesion. Subsequent coculture of these cells synergistically enhanced TFA, which was exclusively dependent on TF molecules that were expressed on EC during coculture. TFA induction required direct contact between monocytes and bacterium-infected EC...
Staphylococcus aureus is isolated from a substantial number of patients with infective endocardit... more Staphylococcus aureus is isolated from a substantial number of patients with infective endocarditis who are not known to have predisposing heart abnormalities. It has been suggested that the infection is initiated by the direct binding of S. aureus to human vascular endothelium. To determine the mutual response of the endothelial cells and the bacteria, we studied the interaction between S. aureus and human vascular endothelium. Scanning electron microscopic analyses showed that binding of S. aureus to human umbilical vein endothelial cells (HUVEC) mainly occurred via thread-like protrusions extending from the cell surface. Bound bacteria appeared to be internalized via retraction of the protrusions into newly formed invaginations of the endothelial cell surface. The growth phase of S. aureus had a major impact on the interaction with HUVEC. Logarithmically growing bacteria showed increased binding to, and were more readily internalized by, HUVEC compared to stationary-phase bacteri...
In the pathogenesis of bacterial endocarditis (BE), the clotting system plays a cardinal role in ... more In the pathogenesis of bacterial endocarditis (BE), the clotting system plays a cardinal role in the formation and maintenance of the endocardial vegetations. The extrinsic pathway is involved in the activation of the coagulation pathway with tissue factor (TF) as the key protein. Staphylococcus aureus is a frequently isolated bacterium from patients with BE. We therefore investigated whether S. aureus can induce TF activity (TFA) on fibrin-adherent monocytes, used as an in vitro model of BE. We also assessed in vivo in rabbits with catheter induced vegetations, the effect of S. aureus infection on vegetational TFA. In vitro experiments showed that adherent S. aureus induced TFA on fibrin-adherent monocytes which was optimal at a bacterium/monocyte ratio of 1 to 1. Monocyte damage occurred when this ratio exceeded 4 to 1 (visually) or 6 to 1 (propidium iodide influx) Consequently, TFA decreased. In vivo S. aureus led to very high bacterial numbers in the vegetations and a significan...
Monocytes are important effector cells in the pathogenesis of bacterial endocarditis since they p... more Monocytes are important effector cells in the pathogenesis of bacterial endocarditis since they provide the tissue factor that activates the coagulation system and maintains established vegetations. Monocytes secrete cytokines that can modulate monocyte tissue factor activity (TFA), thereby affecting the formation and maintenance of vegetations. In this study, we show that monocytes cultured for 4 h on a Streptococcus sanguis -infected fibrin matrix mimicking the in vivo vegetational surface express high levels of TFA. This was accompanied by secretion of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-1α (IL-1α), and IL-1β. After a 24-h incubation period the anti-inflammatory cytokine IL-10 could also be detected. Our data show that, whereas TNF-α and IL-1 have a minor role in the induction of TFA by monocytes cultured on a fibrin matrix, TNF-α but not IL-1 plays an important role in the induction of IL-10 by these cells. In turn, our data show that I...
CD44 variants but when the cells were activated with PMA, expression of exons v3andvBwasdetected.... more CD44 variants but when the cells were activated with PMA, expression of exons v3andvBwasdetected. Conclusions: These data indicate expression of multiple forms of CD44 constitutively on PBMC and suggest that they may have a role to play in normal T-ceil function. However expressfon of v3 and VB was found to be upregulated on Flow Cytometric analysts following PMA stimulation suggesting that, on cellular activation, there may be a conformational change with exposure of epitopes mcognised by these antibodies. Conduslon: TGFB promotes the apoptosis of post-activated T cells and may be a regulatory mechanism acting on extravasated lymphocytes in viva. TGFB may have a role in the microenvimnmental regulation of migratory cells, as it promotes the post-activation induced apoptosis of T cells when added at peak T cell activation. Our results show that TGF/l had no direct effect on T cell activation per se, thereby confirming previous repotts. The potent immunosuppressive and anti-inflammatory effects ascribed to TGFfi may be partly due to the promotion of apoptosis, rather than any direct suppressive effect.
<p>Pups were analysed for chimerism by multiplex PCR and MCA. <b>A</b>) Four of... more <p>Pups were analysed for chimerism by multiplex PCR and MCA. <b>A</b>) Four of the pups derived after transplantation of blastocysts injected with ES cells of clone 9B4 showed presence of the exon 52 deleted <i>hDMD</i> gene (lines 1, 4, 8 and 11). <b>B</b>) Melting curve analysis revealed that all male pups were also chimeric for the <i>mdx</i> point mutation.</p
<p><b>A</b>) Nested PCR revealing exon skipping upon local 51ViM or 53ViM treat... more <p><b>A</b>) Nested PCR revealing exon skipping upon local 51ViM or 53ViM treatment in right and left gastrocnemius and triceps muscle (respectively GR, GL and TR, TL) of two del52hDMD<i>/mdx</i>#35 mice. We confirmed by Sanger sequencing that the upper band in the untreated del52hDMD<i>/mdx</i> samples involves a cryptic splicing event that is occasionally observed in untreated mice of this strain. It contains exon 51, part of intron 51, the last 101 nucleotides of exon 52, exon 53 and multiple stop codons. The arrows indicate the expected heights of fragments lacking exon 51 (700 bp) or exon 53 (721 bp). <b>B</b>) Murine specific nested PCR confirmed that the exon 51 ViM induced low levels of mouse exon 51 skipping. The exon 53 ViM only resulted in exon 53 skipping in the human transcript as no skipping band was seen in the PCR performed with mouse-specific primers (expected size 483 bp). Sanger sequence confirmed that the smaller fragment obtained in the ViM exon 51 treated muscles contained the boundary of exon 50–52. Human ctrl; healthy human control sample. The arrow indicates the expected height of fragments lacking exon 51 (463 bp). <b>C-D</b>) Exon skipping resulted in the restored dystrophin expression in gastrocnemius (C) and triceps (D) of 51ViM and 53ViM treated mice.</p
<p><b>A</b>) Western blot analyses of heart and quadriceps, incubated with eith... more <p><b>A</b>) Western blot analyses of heart and quadriceps, incubated with either GTX (human and mouse specific) or Mandys106 (human specific). Wild type expression levels of human dystrophin were observed in hDMD/<i>mdx</i> mice. Notably, del52hDMD/<i>mdx</i>#37 mice expressed traces of human dystrophin, in both cardiac and skeletal muscle, while this was not observed in del52hDMD/<i>mdx</i>#35 and <i>mdx</i>(BL6) mice. <b>B</b>) Sections of the heart and quadriceps stained with human specific dystrophin antibodies. Expression of human dystrophin is at wild type level in hDMD/<i>mdx</i> mice as anticipated. Both C57BL/6J, <i>mdx</i>(BL6) and del52hDMD/<i>mdx</i>#35 mice did not express human dystrophin. Interestingly, in most fibers of del52hDMD/<i>mdx</i>#37 mice, human dystrophin was expressed at low levels. Haematoxylin and eosin staining revealed signs of degeneration and regeneration in the quadriceps of both del52hDMD/<i>mdx</i> strains, as evident by variation in fiber size, centralized nuclei and patches of fibrosis and inflammation. Overall pathology appeared to be slightly less extensive in del52hDMD/<i>mdx</i>#37 mice compared to <i>mdx</i>(BL6) and del52hDMD/<i>mdx</i>#35 mice. <b>C</b>) Almost no centralized nuclei were found in wild type mice, while half of the myofibers in <i>mdx</i>(BL6) and del52hDMD/<i>mdx</i>#35 mice had centrally located nuclei. The percentage in del52hDMD/<i>mdx</i>#37 mice was with 26% significantly lower. Data were based on manual counts of 5 randomly taken pictures of 2 males and 2 females per genotype. Asterisks indicate <i>P</i><0.01.</p
<p><b>A</b>) Forelimb grip strength was impaired in <i>mdx</i>(BL6)... more <p><b>A</b>) Forelimb grip strength was impaired in <i>mdx</i>(BL6) and del52hDMD<i>/mdx</i> mice. <b>B</b>) hDMD/<i>mdx</i> mice were resistant against fatigue, while muscles of <i>mdx</i>(BL6) and del52hDMD<i>/mdx</i> mice were fatigued at the end of the grip strength protocol. Performance in hanging tests starting with two <b>C</b>) and four limbs <b>D</b>) was impaired in <i>mdx</i>(BL6) and del52hDMD<i>/mdx</i> mice. Overall, del52hDMD<i>/mdx</i>#37 mice outperformed <i>mdx</i>(BL6) and del52hDMD<i>/mdx</i>#35 mice. <b>E</b>) Creatine kinase levels were elevated in both del52hDMD<i>/mdx</i> and <i>mdx</i>(BL6) strains compared to hDMD/<i>mdx</i> mice. Asterisk indicates <i>P</i><0.05, data are represented as the mean ± SEM. hDMD/<i>mdx</i>, <i>mdx</i>(BL6) and del52hDMD<i>/mdx</i>#35 groups consisted of n = 3 males and n = 2 females (one male <i>mdx</i>(BL6) mouse died), while the del52hDMD<i>/mdx</i>#37 group consisted of n = 2 males and n = 3 females.</p
<p>Single ES clones were cultured in 96-well plates and DNA was isolated and used as templa... more <p>Single ES clones were cultured in 96-well plates and DNA was isolated and used as template in a multiplex PCR. Here the exons 46, 51 and 52 of the <i>hDMD</i> gene were analysed where exon 46 and 51 are positive controls and exon 52 the target to be deleted. <b>A</b>) An example is shown where candidate samples 2 and 5 are of interest because they are negative for exon 52 but positive for the control exons. <b>B</b>) For a large number of clones additional fragments were found for exon 52, suggesting non-homologues end joining (NHEJ) of TALEN induced double stranded breaks <b>C</b>) Representative image of LR-PCR performed on DNA of sub-clones of four exon 52 negative clones (9B4, 10H2, 11C9 and 11E7). LR-PCR was performed with primers targeting intron 51 (outside the targeting arm) and blasticidin (only present after homologous recombination), to rule out loss of PCR primer recognition sites by NHEJ and to confirm true targeting. <b>D</b>) RT-PCR was performed for RNA isolated from embryoid bodies of selected clones. The different fragments were isolated, purified and Sanger sequence analysed. In the wild type situation exon 52 was present, whereas in the properly targeted clones exon 52 was not present. This confirmed the exon 52 deletion on RNA level.</p
Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease generally caused by reading ... more Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease generally caused by reading frame disrupting mutations in the DMD gene resulting in loss of functional dystrophin protein. The reading frame can be restored by antisense oligonucleotide (AON)-mediated exon skipping, allowing production of internally deleted, but partially functional dystrophin proteins as found in the less severe Becker muscular dystrophy. Due to genetic variation between species, mouse models with mutations in the murine genes are of limited use to test and further optimize human specific AONs in vivo. To address this we have generated the del52hDMD/mdx mouse. This model carries both murine and human DMD genes. However, mouse dystrophin expression is abolished due to a stop mutation in exon 23, while the expression of human dystrophin is abolished due to a deletion of exon 52. The del52hDMD/mdx model, like mdx, shows signs of muscle dystrophy on a histological level and phenotypically mild functional impairment. Local administration of human specific vivo morpholinos induces exon skipping and dystrophin restoration in these mice. Depending on the number of mismatches, occasional skipping of the murine Dmd gene, albeit at low levels, could be observed. Unlike previous models, the del52hDMD/mdx model enables the in vivo analysis of human specific AONs targeting exon 51 or exon 53 on RNA and protein level and muscle quality and function. Therefore, it will be a valuable tool for optimizing human specific AONs and genome editing approaches for DMD.
Duchenne muscular dystrophy (DMD) is a muscle-wasting disease in which muscle is continuously dam... more Duchenne muscular dystrophy (DMD) is a muscle-wasting disease in which muscle is continuously damaged, resulting in loss of muscle tissue and function. Antisense-mediated exon skipping is a promising therapeutic approach for DMD. This method uses sequence specific antisense oligonucleotides (AONs) to reframe disrupted dystrophin transcripts. As AONs function in a sequence specific manner, human specific AONs cannot be tested in the mdx mouse, which carries a mutation in the murine Dmd gene. We have previously generated a mouse model carrying the complete human DMD gene (hDMD mouse) integrated in the mouse genome to overcome this problem. However, as this is not a disease model, it cannot be used to study the effect of AON treatment on protein level and muscle function. Therefore, our long term goal is to generate deletions in the human DMD gene in a mouse carrying the hDMD gene in an mdx background. Towards this aim, we generated a male ES cell line carrying the hDMD gene while having the mdx point mutation. Inheritance of the hDMD gene by the ES cell was confirmed both on DNA and mRNA level. Quality control of the ES cells revealed that the pluripotency marker genes Oct-4 and Nanog are well expressed and that 85% of cells have 40 chromosomes. Germ line competence of this cell line has been confirmed, and 2 mice strains were derived from this cell line and crossed back on a C57BL6 background: hDMD/mdx and mdx(BL6). Funding Statement MV and LV are paid by a grant from the Prinses Beatrix Spierfonds (the Netherlands) and AAR receives funding from ZonMw, the Prinses Beatrix Spierfonds and the Duchenne Parent Project (the Netherlands). The infrastructure of the Center for Medical Systems Biology and the Center for Biomedical Genetics was utilized to conduct the reported work. JK, JC, AAR, and JV report being employed by LUMC, which has patents on exon skipping. As a co-author on some of these patents AAR is entitled to a share of royalties. MV, JK, JC, LV and JV declare no conflict of interest. Research groups are exploring various therapies to slow down disease progression. Therapies are mainly based on restoration of dystrophin expression and on improving muscle quality. Antisense-mediated exon skipping, aiming to convert a severe DMD phenotype into a milder Becker muscular dystrophy phenotype is currently closest to clinical application 5. In DMD patients the reading-frame of the dystrophin mRNA is disrupted resulting in a prematurely truncated, non functional dystrophin protein. Utilizing antisense oligonucleotides (AONs), the splicing of an out of frame exon during maturation of the pre-mRNA is prevented to restore the reading frame, allowing the production of an internally deleted but partially functional dystrophin protein. AONs target exons in a sequence specific manner and therefore human specific AONs cannot be tested in mouse models 6. Consequently, the mdx mouse, which carries a mutation in exon 23 of the murine Dmd gene and is the most widely used model for DMD research, cannot be used as a preclinical model to test human specific AONs. Therefore we have developed the hDMD mouse 7 , which carries the complete human DMD (hDMD) gene integrated in the mouse genome (B6.DBA2.129-hDMD tg/tg , from now on referred to as hDMD mouse). When crossed with mdx mice, the human dystrophin can compensate for the lack of mouse dystrophin and prevent pathology 7. A drawback of the hDMD mouse is that it is not a disease model, due to its expression of human dystrophin. This makes it impossible to study the restoration of human dystrophin and muscle function and quality upon AON treatment. A mouse model to allow in vivo assessment of human specific AONs would be very valuable. Preferably this model would not express mouse dystrophin and would have a deletion in the human DMD gene (?hDMD) in the mutation hotspot region (between exon 45 and
Rare diseases can be caused by genetic mutations that disrupt normal pre-mRNA splicing. Antisense... more Rare diseases can be caused by genetic mutations that disrupt normal pre-mRNA splicing. Antisense oligonucleotide treatment to the splicing thus has therapeutic potential for many rare diseases. In this review we will focus on the state of the art on exon skipping using antisense oligonucleotides as a potential therapy for rare genetic diseases, outlining how this versatile approach can be exploited to correct for different mutations.
Motivation: Advances in sequencing technologies and computational algorithms have enabled the stu... more Motivation: Advances in sequencing technologies and computational algorithms have enabled the study of genomic variants to dissect their functional consequence. Despite this unprecedented progress, current tools fail to reliably detect and characterize more complex allelic variants, such as short tandem repeats (STRs). We developed TSSV as an efficient and sensitive tool to specifically profile all allelic variants present in targeted loci. Based on its design, requiring only two short flanking sequences, TSSV can work without the use of a complete reference sequence to reliably profile highly polymorphic, repetitive or uncharacterized regions. Results: We show that TSSV can accurately determine allelic STR structures in mixtures with 10% representation of minor alleles or complex mixtures in which a single STR allele is shared. Furthermore, we show the universal utility of TSSV in two other independent studies: characterizing de novo mutations introduced by transcription activator-like effector nucleases (TALENs) and profiling the noise and systematic errors in an IonTorrent sequencing experiment. TSSV complements the existing tools by aiding the study of highly polymorphic and complex regions and provides a high-resolution map that can be used in a wide range of applications, from personal genomics to forensic analysis and clinical diagnostics. Availability and implementation: We have implemented TSSV as a Python package that can be installed through the command-line using pip install TSSV command. Its source code and documentation are
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