Assessing toxicity of polyamidoamine dendrimers by neuronal signaling functions

We report for the first time on neuronal signaling for the evaluation of interactions between native plasmamembrane and polyamidoamine (PAMAM) dendrimers. Generation 5 polycationic (G5-NH(2)), novel β-D-glucopyranose-conjugated G5-NH(2) and generation 4.5 polyanionic (G4.5-COONa) polyamidoamine (PAMAM) dendrimers (1-0.0001 mg/ml) were applied in acute brain slices. Functional toxicity assessments-validated by fluorescence imaging of dead cells-were performed by employing electrophysiological indicators of plasma membrane breakdown and synaptic transmission relapse. Irreversible membrane depolarization and decrease of membrane resistance predicted substantial functional neurotoxicity of unmodified G5-NH(2), but not of the G4.5-COONa PAMAM dendrimers. Model calculations suggested that freely moving protonated NH(2) groups of terminal monomeric units of PAMAM dendrimers may be able directly destroy the membrane or inhibit important K(+) channel function via contacting the positively charged NH(2). In accordance, conjugation of surface amino groups by β-D-glucopyranose units reduced functional neurotoxicity that may hold great potential for biomedical applications.     

Peritubular capillary basement membrane changes in chronic renal allograft rejection: Comparison of light microscopic and ultrastructural observations

Marked peritubular capillary basement membrane (PTCBM) multilayering, the ultrastructural feature of chronic antibody-mediated rejection (ABMR) of kidney allografts, was found to correspond histologically to PTCs with thickened BMs; such PTCs have been suggested as a novel histological marker of chronic rejection. We investigated whether scoring of PTCBM thickening can substitute the ultrastructural search for PTCBM multilayering. The thickening was graded in PAS- and Jones-stained sections in 110 biopsies from recipients with a late dysfunction, all examined ultrastructurally for transplant capillaropathy (≥3 PTCs with ≥5 BM layers). Grade 0 indicated no thickening. Grade 1 and grade 2 were assigned when the PTCBMs were as thick as or thicker than those of the non-atrophic tubules, and duplication/chain-like lamination of the PTCBM was noted in ≤3 or ≥4 high-power fields, respectively. The series was enrolled in subgroups of those with and those without histopathological lesions of chronic rejection. Fifty-six biopsies displayed lesions of chronic ABMR. Transplant capillaropathy was demonstrated in 40 biopsies. Grade 2 thickening furnished a substantial interobserver concordance rate (κ = 0.803) and correlated with the transplant capillaropathy. Jones staining performed somewhat better in scoring than PAS staining. Grade 2 thickening was verified in 35 biopsies involving chronic ABMR, and in one control biopsy (sensitivity 61.4%, specificity 0.98). Grade 1 thickening was not suggestive of chronic ABMR at all. In conclusion, grade 2 thickening can be regarded as the histopathological lesion of chronic ABMR; however, electron microscopy remains the gold standard in the assessment of PTCBM changes.     

Rapid detection of myocardial infarction with a sensitive troponin test

Rapid identification and treatment of patients with a myocardial infarction (MI) is mandatory. We studied the diagnostic capacities of a sensitive troponin assay for detection of MI in emergency department patients within 2 hours after arrival. The study included 157 patients suspected of having non-ST-elevation acute coronary syndrome. Blood was drawn on arrival (T0) and 2 (T2), 6, and 12 hours later. At T2, a troponin concentration above the MI cutoff is 87% sensitive and 100% specific for MI detection (positive predictive value [PPV], 100%; negative predictive value [NPV], 96%). If a difference of more than 30% between the troponin measurements at T0 and T2 in the absence of an absolute troponin increase above the 99th percentile of a reference population is also considered indicative of MI, the sensitivity increases to 100% and specificity decreases to 87% (PPV, 70%; NPV, 100%). Sensitivity and specificity of creatine kinase-MB and myoglobin are lower than those of troponin. By using a sensitive troponin assay and simple algorithms, the diagnosis of MI can be determined within 2 hours after arrival at the emergency department. Measurement of myoglobin and creatine kinase-MB has no added value.     

Retrospective diagnosis of X-linked hyper-IgM syndrome in a family with multiple deaths of affected males

All males in two generations of a Hungarian family died of interstitial pneumonia. History and records suggested X-linked hyper-IgM syndrome (X-HIGM). DNA sequencing of a female carrier revealed a c. 654C->A transversion of the CD40L gene that predicts premature termination of CD40L synthesis. This report points to the importance of early carrier detection and genetic counseling in families with X-linked primary immunodeficiency diseases. We propose that the c.654C->A sequence variant may associate with severe X-HIGM phenotype.     

Laryngeal mask airway is associated with an increased incidence of adverse respiratory events in children with recent upper respiratory tract infections

BACKGROUND: The laryngeal mask airway (LMA) has been advocated as an alternative technique to tracheal intubation for airway management of children with recent upper respiratory tract infections (URIs). The authors determined the occurrence of adverse respiratory events and identified the associated risk factors to assess the safety of LMA in children. METHODS: During a period of 5 months, parents of children scheduled to undergo general anesthesia with an LMA were asked to fill out a questionnaire regarding their child's medical history and potential symptoms of URI. In addition, all episodes of adverse respiratory events in the perioperative period (laryngospasm, bronchospasm, coughing, airway obstruction, and oxygen desaturation) as well as details of anesthesia management were recorded. RESULTS: Among the 831 children included in the study, 27% presented with a history of a recent URI within the last 2 weeks before anesthesia. The presence of a recent URI doubled the incidence of laryngospasm (odds ratio, 2.6; 95% confidence interval, 1.3-5.0), coughing (odds ratio, 2.7; 95% confidence interval, 1.7-4.3), and oxygen desaturation (odds ratio, 1.9; 95% confidence interval, 1.2-2.8). This incidence was even higher in young children; in those undergoing ear, nose, and throat surgery; and when there were multiple attempts to insert the LMA. CONCLUSION: An LMA used in children with recent URIs was associated with a higher incidence of laryngospasm, cough, and oxygen desaturation compared with healthy children. However, the overall incidence of adverse respiratory events was low, suggesting that if anesthesiologists allow at least a 2-week interval after a URI, they can safely proceed with anesthesia using an LMA.     

Peroxidasin Is Secreted and Incorporated into the Extracellular Matrix of Myofibroblasts and Fibrotic Kidney

Mammalian peroxidases are heme-containing enzymes that serve diverse biological roles, such as host defense and hormone biosynthesis. A mammalian homolog of Drosophila peroxidasin belongs to the peroxidase family; however, its function is currently unknown. In this study, we show that peroxidasin is present in the endoplasmic reticulum of human primary pulmonary and dermal fibroblasts, and the expression of this protein is increased during transforming growth factor-β1-induced myofibroblast differentiation. Myofibroblasts secrete peroxidasin into the extracellular space where it becomes organized into a fibril-like network and colocalizes with fibronectin, thus helping to form the extracellular matrix. We also demonstrate that peroxidasin expression is increased in a murine model of kidney fibrosis and that peroxidasin localizes to the peritubular space in fibrotic kidneys. In addition, we show that this novel pathway of extracellular matrix formation is unlikely mediated by the peroxidase activity of the protein. Our data indicate that peroxidasin secretion represents a previously unknown pathway in extracellular matrix formation with a potentially important role in the physiological and pathological fibrogenic response.Peroxidases are heme-containing enzymes with highly conserved structure, serving diverse functions in the plant and animal kingdom.¹ Peroxidases catalyze the oxidation of various substrates in the presence of H₂O₂. Mammalian peroxidases have an important role in several physiological processes including host defense and hormone biosynthesis. The family of mammalian peroxidases consists of myeloperoxidase, eosinophil peroxidase, lactoperoxidase, thyroid peroxidase, and the mammalian peroxidasin. Myeloperoxidase, eosinophil peroxidase, and lactoperoxidase have antimicrobial activity and serve in the first line of host defense, while thyroid peroxidase has an essential role in the biosynthesis of thyroid hormones.^2,3,4 The function of the mammalian peroxidasin is currently unknown. Peroxidases in plants and in lower animal species frequently participate in extracellular matrix (ECM) formation. In the presence of H₂O₂, peroxidases enzymatically cross-link extracellular proteins through tyrosine residues.⁵ ECM stabilization by dityrosine bridges is well-documented during sea urchin fertilization, where secreted ovoperoxidase is responsible for the formation of cross-links.⁶ Dityrosine formation is also involved in the stabilization of C. elegans cuticle, where dual oxidases, carrying both NADPH oxidase and peroxidase-like domains, provide hydrogen peroxide for the crosslinking reaction.⁷Peroxidasin (PXDN), a unique form of peroxidase was first identified in Drosophila melanogaster.⁸ Beside containing a peroxidase domain, which is highly homologous to other animal peroxidases, peroxidasin also contains protein domains characteristic for proteins of the ECM. Drosophila PXDN was found to be expressed in several stages of development, but the exact function remained unknown.⁸ Little is still known about the mammalian PXDN protein. A human homolog of Drosophila PXDN was originally identified as a p53-responsive gene product from a colon cancer cell line, but it was not characterized in detail.⁹ An independent cloning effort, using subtractive hybridization also led to the identification of the mammalian PXDN gene, which was originally named melanoma gene 50, based on the expression in melanoma samples.¹⁰ This latter study has characterized PXDN as a possible potent melanoma-associated antigen, but it did not examine the possible physiological role of the protein.Here we demonstrate that peroxidasin is expressed by human primary cells, including fibroblasts of different origin, where the protein is localized to the endoplasmic reticulum. On stimulation by transforming growth factor (TGF)-β1, differentiating myofibroblasts show increased expression of peroxidasin. The protein becomes secreted to the extracellular space where it is organized into a fibril-like network. We also show that this pathway of ECM formation is probably not mediated by the peroxidase activity of the protein. Our results suggest that beside the secretion of well-known constituents of the ECM, PXDN secretion by myofibroblasts is a novel way of ECM modification in wound repair and tissue fibrosis.     

Four cases of disseminated Mycobacterium bovis infection following intravesical BCG instillation for treatment of bladder carcinoma

Intravesical BCG (bacillus Calmette-Guérin) instillation is a first-line treatment for superficial transitional cell carcinoma of the bladder. A rare but severe complication of BCG immunotherapy is the development of disseminated BCG disease, which can result in miliary pneumonitis, granulomatous hepatitis, soft tissue infections, bone marrow involvement, and sepsis. Symptoms can present as early as a few hours or as late as several months following the BCG therapy. The key finding in disseminated BCG disease is the formation of caseating granulomas in distant organs; detection of BCG organisms from tissue samples can be difficult. Recommended treatment for disseminated BCG disease includes a combination of antituberculous medications (with the exception of pyrazinamide, to which BCG is typically resistant) and a tapering course of steroids. We present the cases of four patients who developed granulomatous infection consistent with disseminated disease after intravesical BCG treatment and provide a summary of current clinical management recommendations.