Phadiatop Infant in the Diagnosis of Atopy in Children with Allergy-Like Symptoms

Background and Objective. Allergy-like symptoms such as wheezing and eczema are common in young children and an early diagnosis is important to initiate correct management. The objective of this study was to evaluate the diagnostic performance of Phadiatop Infant, an in vitro test for determination of early sensitisation to food and inhalant allergens. Patients and Methods. The study was conducted, retrospectively, using frozen sera from 122 children (median age 2.7 years) admitted to the hospital with suspected allergic symptoms. The doctor's diagnosis atopic/nonatopic was based on routinely used procedures such as clinical evaluation, SPT, total and allergen-specific IgE antibodies. The performance of Phadiatop Infant was evaluated in a blinded manner against this diagnosis. Results. Eighty-four of the 86 children classified as atopic showed a positive Phadiatop Infant test. Thirty-six were classified as nonatopic, 32 of who had a negative test. With a prevalence of atopy of 70% in this population, this gives a sensitivity of 98%, a specificity of 89%, and a positive and negative predictive value of 95% and 94%, respectively. Conclusion. The results from the present study suggest that Phadiatop Infant could be recommended as a complement to the clinical information in the differential diagnosis on IgE-mediated disease in young children with allergy-like symptoms

Tissue distribution and transcriptional regulation of CCN5 in the heart after myocardial infarction

Abstract CCN5 is a divergent member of the cellular communication network factor (CCN) family in that it lacks the carboxyl terminal cystine knot domain common to the other CCN family members. CCN5 has been reported to antagonize the profibrotic actions of CCN2 and to inhibit myocardial collagen deposition and fibrosis in chronic pressure overload of the heart. However, what mechanisms that regulate CCN5 activity in the heart remain unknown. Recombinant, replication defective adenovirus encoding firefly luciferase under control of the human CCN5 promoter was prepared and used to investigate what mechanisms regulate CCN5 transcription in relevant cells. Tissue distribution of CCN5 in hearts from healthy mice and from mice subjected to myocardial infarction was investigated. Contrary to the profibrotic immediate early gene CCN2, we find that CCN5 is induced in the late proliferation and maturation phases of scar healing. CCN5 was identified principally in endothelial cells, fibroblasts, smooth muscle cells, and macrophages. Our data show that CCN5 gene transcription and protein levels are induced by catecholamines via β 2 -adrenergic receptors. Myocardial induction of CCN5 was further confirmed in isoproterenol-infused mice. We also find that CCN5 transcription is repressed by TNF-α, an inflammatory mediator highly elevated in early phases of wound healing following myocardial infarction. In conclusion, CCN5 predominates in endothelial cells, fibroblasts, and macrophages of the differentiating scar tissue and its transcription is conversely regulated by β 2 -adrenergic agonists and TNF-α

Connective tissue growth factor (CCN2) is a matricellular preproprotein controlled by proteolytic activation.

Connective tissue growth factor (CTGF; now often referred to as CCN2) is a secreted protein predominantly expressed during development, in various pathological conditions that involve enhanced fibrogenesis and tissue fibrosis, and in several cancers and is currently an emerging target in several early-phase clinical trials. Tissues containing high CCN2 activities often display smaller degradation products of full-length CCN2 (FL-CCN2). Interpretation of these observations is complicated by the fact that a uniform protein structure that defines biologically active CCN2 has not yet been resolved. Here, using DG44 CHO cells engineered to produce and secrete FL-CCN2 and cell signaling and cell physiological activity assays, we demonstrate that FL-CCN2 is itself an inactive precursor and that a proteolytic fragment comprising domains III (thrombospondin type 1 repeat) and IV (cystine knot) appears to convey all biologically relevant activities of CCN2. In congruence with these findings, purified FL-CCN2 could be cleaved and activated following incubation with matrix metalloproteinase activities. Furthermore, the C-terminal fragment of CCN2 (domains III and IV) also formed homodimers that were ∼20-fold more potent than the monomeric form in activating intracellular phosphokinase cascades. The homodimer elicited activation of fibroblast migration, stimulated assembly of focal adhesion complexes, enhanced RANKL-induced osteoclast differentiation of RAW264.7 cells, and promoted mammosphere formation of MCF-7 mammary cancer cells. In conclusion, CCN2 is synthesized and secreted as a preproprotein that is autoinhibited by its two N-terminal domains and requires proteolytic processing and homodimerization to become fully biologically active

CTGF/CCN2 Postconditioning Increases Tolerance of Murine Hearts towards Ischemia-Reperfusion Injury

<div><p>Background and Purpose</p><p>Previous studies of ischemia-reperfusion injury (IRI) in hearts from mice with cardiac-restricted overexpression of CCN2 have shown that CCN2 increases tolerance towards IRI. The objectives of this study were to investigate to what extent post-ischemic administration of recombinant human CCN2 (rhCCN2) would limit infarct size and improve functional recovery and what signaling pathways are involved.</p><p>Experimental Approach</p><p>Isolated mice hearts were perfused ad modum Langendorff, subjected to no-flow, global ischemia, and subsequently, exposed to mammalian cell derived, full-length (38-40kDa) rhCCN2 (250 nM) or vehicle during the first 15 min of a 60 min reperfusion period.</p><p>Key Results</p><p>Post-ischemic administration of rhCCN2 resulted in attenuation of infarct size from 58 ± 4% to 34 ± 2% (p < 0.001) which was abrogated by concomitant administration of the PI3 kinase inhibitor LY294002 (45 ± 3% vs. 50 ± 3%, ns). In congruence with reduction of infarct size rhCCN2 also improved recovery of left ventricular developed pressure (p < 0.05). Western blot analyses of extracts of ex vivo-perfused murine hearts also revealed that rhCCN2 evoked concentration-dependent increase of cardiac phospho-GSK3β (serine-9) contents.</p><p>Conclusions and Implications</p><p>We demonstrate that post-ischemic administration of rhCCN2 increases the tolerance of ex vivo-perfused murine hearts to IRI. Mechanistically, this postconditioning effect of rhCCN2 appeared to be mediated by activation of the reperfusion injury salvage kinase pathway as demonstrated by sensitivity to PI3 kinase inhibition and increased CCN2-induced phosphorylation of GSK3β (Ser-9). Thus, the rationale for testing rhCCN2-mediated post-ischemic conditioning of the heart in more complex models is established.</p></div

Schematic of experimental protocols.

<p>A. For phosphoprotein studies hearts were perfused with Krebs Henseleit buffer at 37°C in Langendorff setups. The perfused hearts were allowed to stabilize for 5 min before perfusion for 15 min without (Ctrl p) or with rhCCN2 (CCN2 p; 250 nM). B and C. For the ischemia-reperfusion studies Langendorff-perfused hearts were allowed to stabilize for 25 min before start of no-flow global ischemia. No-flow ischemia was maintained for 40 min (Ctrl 40i, CCN2 40i, Ctrl 40i +LY and CCN2 40i +LY) or 25 min (Ctrl 25i and CCN2 25i). In the CCN2 40i, CCN2 40i + LY and CCN2 25i the first 15 min of the 60 min reperfusion were in the presence of 250nM recombinant human CCN2. In the Ctrl 40i + LY and CCN2 40i + LY the entire perfusion was performed in the presence of 50μM LY294002.</p

Infarct sizes in hearts subjected to rhCCN2 postconditioning.

<p>A: Morphometric analysis of infarct sizes as percentages of total heart slice area. B: Representative example of TTC-stained hearts from the groups Ctrl 40i and CCN2 40i. Data are mean ± SEM of N = 11 in each group. * <i>P</i><0.05</p

Phospho-GSK3β (Ser 9) contents in rhCCN2 stimulated hearts.

<p>Western blot analysis of phospho-GSK3β (Ser-9) contents in extracts of murine hearts following ex vivo perfusion of the hearts (Langendorff-perfusion) in the absence or presence of increasing concentrations of recombinant human CCN2. Each lane on the Western blot represents tissue from individual hearts. The histogram demonstrates group means of phospho-GSK3β contents assessed by densitometric analysis of the immunoreactive bands on the Western blot. The phospho-GSK3β (Ser-9) contents are displayed relative to total immunoreactive GSK3β contents. (Each group is mean of 2–3 <i>ex vivo</i>-perfused hearts)</p

Cardiac function in hearts subjected to rhCCN2 postconditioning.

<p>Cardiac function of hearts subjected to 25 min of ischemia and subsequently perfused in the absence or presence of rhCCN2 (250 nM) during the first 15 min of reperfusion. The figure demonstrates recording of heart rate (A), left ventricular end-systolic pressure, LVSP (B), left ventricular end-diastolic pressure (LVEDP) (C), and left ventricular developed pressure, LVDP (LVSP—LVEDP) (D) at baseline and during reperfusion after ischemia. LVEDP (C), illustrates the “ischemic contracture” of the Langendorff global ischemia system during ischemia. Recovery of LVDP is significantly increased in the group receiving recombinant human CCN2 during reperfusion (CCN2 25i). Data are mean, ± SEM for the first and last data point of N = 10 in each group. * <i>P</i><0.05.</p