Christof Meischl

Genetic analysis of patients with leukocyte adhesion deficiency: genomic sequencing reveals otherwise undetectable mutations.

OBJECTIVE The aim of this study was to analyze mutations in DNA from patients with leukocyte adhesion deficiency (LAD), an immunodeficiency caused by absence of the beta(2) subunit (CD18) of the leukocyte integrins LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), p150,95 (CD11c/CD18), and CR4 (CD11d/CD18). METHODS We developed genomic DNA PCR sequencing to detect mutations not only in exons but also in introns. RESULTS Eight LAD patients were analyzed, of which five had homozygous mutations, i.e., a 0.8-kb deletion, a branchpoint mutation in intron 5 causing mRNA missplicing, a nonsense mutation, and two missense mutations. Four of these mutations are novel. We cotransfected the two mutant CD18 proteins with normal CD11a, b, or c in COS cells. This resulted in absence of all three beta(2) integrins on the surface of cells transfected with CD18(252Arg). However, CD18(593Cys) supported some LFA-1 and p150,95 formation in COS cells. The other three patients were compound heterozygotes in which only one allele had previously been characterized, because the other alleles were undetectable at the cDNA level. We identified the unknown mutations as a novel two-nucleotide deletion, a nonsense mutation, and a single nucleotide deletion. CONCLUSION Our method allows identification of mutations in CD18 from genomic DNA. This opens the possibility of early prenatal diagnosis of LAD and reliable carrier detection.

Inhibition of sPLA2-IIA, C-reactive Protein or Complement: New Therapy for Patients with Acute Myocardial Infarction?

Reperfusion of ischemic myocardium after acute myocardial infarction (AMI) induces a local activation of inflammatory reactions that results in ischemia/reperfusion (I/R)-injury. I/R-injury contributes considerably to the total cell damage in the heart after AMI. Secretory phospolipase A2-IIA (sPLA2-IIA), C-reactive protein (CRP) and complement are inflammatory mediators that have been demonstrated to play key roles in I/R injury. From studies by us and others a mechanism emerged in which sPLA2-IIA binds to reversibly damaged cardiomyocytes and subsequently induces cell death, partly by potentiating binding of CRP and subsequent complement activation. Next to this, sPLA2-IIA also has a direct toxic effect, independent of CRP or complement. Therefore, these studies indicate a crucial role of inflammatory mediators in ischemia/reperfusion injury. This review will focus on the pathogenic effects of sPLA2-IIA, CRP and complement and on the putative therapeutic effects of inhibitors of these inflammatory mediators in acute myocardial infarction.

H9c2 cardiomyoblasts produce thyroid hormone

Thyroid hormone acts on a wide range of tissues. In the cardiovascular system, thyroid hormone is an important regulator of cardiac function and cardiovascular hemodynamics. Although some early reports in the literature suggested an unknown extrathyroidal source of thyroid hormone, it is currently thought to be produced exclusively in the thyroid gland, a highly specialized organ with the sole function of generating, storing, and secreting thyroid hormone. Whereas most of the proteins necessary for thyroid hormone synthesis are thought to be expressed exclusively in the thyroid gland, we now have found evidence that all of these proteins, i.e., thyroglobulin, DUOX1, DUOX2, the sodium-iodide symporter, pendrin, thyroid peroxidase, and thyroid-stimulating hormone receptor, are also expressed in cardiomyocytes. Furthermore, we found thyroglobulin to be transiently upregulated in an in vitro model of ischemia. When performing these experiments in the presence of 125 I, we found that 125 I was integrated into thyroglobulin and that under ischemia-like conditions the radioactive signal in thyroglobulin was reduced. Concomitantly we observed an increase of intracellularly produced, 125 I-labeled thyroid hormone. In conclusion, our findings demonstrate for the first time that cardiomyocytes produce thyroid hormone in a manner adapted to the cells environment.

NOX2, p22phox and p47phox are targeted to the nuclear pore complex in ischemic cardiomyocytes colocalizing with local reactive oxygen species

Background: NADPH oxidases play an essential role in reactive oxygen species (ROS)-based signaling in the heart. Previously, we have demonstrated that (peri)nuclear expression of the catalytic NADPH oxidase subunit NOX2 in stressed cardiomyocytes, e.g. under ischemia or high concentrations of homocysteine, is an important step in the induction of apoptosis in these cells. Here this ischemia-induced nuclear targeting and activation of NOX2 was specified in cardiomyocytes. Methods: The effect of ischemia, mimicked by metabolic inhibition, on nuclear localization of NOX2 and the NADPH oxidase subunits p22phox and p47phox, was analyzed in rat neonatal cardiomyoblasts (H9c2 cells) using Western blot, immuno-electron microscopy and digital-imaging microscopy. Results: NOX2 expression significantly increased in nuclear fractions of ischemic H9c2 cells. In addition, in these cells NOX2 was found to colocalize in the nuclear envelope with nuclear pore complexes, p22phox, p47phox and nitrotyrosine residues, a marker for the generation of ROS. Inhibition of NADPH oxidase activity, with apocynin and DPI, significantly reduced (peri)nuclear expression of nitrotyrosine. Conclusion: We for the first time show that NOX2, p22phox and p47phox are targeted to and produce ROS at the nuclear pore complex in ischemic cardiomyocytes.

Initial characterization of C16orf89, a novel thyroid-specific gene.

BACKGROUND Thyroid hormone is prerequisite for proper fetal and postnatal neurodevelopment, growth, and metabolism. Although much progress has been made in the characterization of genes implicated in thyroid development and function, the majority of genes involved in this process are still unknown. We have previously applied serial analysis of gene expression (SAGE) to identify novel genes preferentially expressed in the thyroid, and this has resulted in the characterization of DUOX2 and IYD (also known as DEHAL1), two genes encoding essential enzymes in the production of thyroid hormone. In the current study we characterize the gene C16orf89, which is linked to another thyroid-specific SAGE tag CCAGCTGCCT. METHODS We establish tissue-specific expression of C16orf89 using novel tissue-specific SAGE libraries and quantitative polymerase chain reaction. In addition, we characterize the C16orf89 gene and protein, and analyze its mRNA expression in response to thyrotropin and during mouse development. RESULTS C16orf89 is predominantly expressed in human thyroid tissue with a specificity intermediate between thyroid transcription factors and proteins involved in thyroid hormone synthesis. C16orf89 shows the same expression pattern as Nkx2-1 (thyroid transcription factor 1) from embryonic day (E) 17.5 onward in the developing mouse thyroid and lung. The developmental timing of C16orf89 mRNA expression is similar to that of the iodide transporter Slc5a5 (also known as Nis). Both transcripts are detected from E17.5 in the developing thyroid. This is clearly later than the onset of Tg mRNA expression (from E14.5), while Nkx2-1 and Iyd mRNA can already be detected in the E12.5 thyroid. In in vitro cell culture C16orf89 expression is stimulated by thyrotropin. The major splice variant encodes a 361 amino acid protein that is well conserved between mammals, contains an N-terminal signal peptide, is secreted in a glycosylated form, and does not contain any known functional domain. CONCLUSIONS We present a novel gene highly expressed in thyroid that encodes a currently enigmatic protein.