Angioedema: A potential complication of dupilumab in atopic dermatitis.

Dupilumab is a relatively new and quite effective medication for recalcitrant atopic dermatitis in patients over 6 years of age. Here, we present a 12-year-old girl with progressively worsening episodic facial swelling and erythema while treated with dupilumab. This case highlights the possibility of angioedema as an adverse effect of dupilumab treatment.

Electrochemical determination of heme-linked pKa values and the importance of using fluoride binding in heme proteins.

The ultraviolet-visible (UV-vis) spectroelectrochemical measurements of heme proteins in the presence of a heme-bound fluoride ion can be used as a probe for heme-linked ionizations of acid-base groups in the heme pocket. A detailed study of the pH dependence of the midpoint potential of skeletal horse myoglobin (Mb) with a heme-bound fluoride ion (Mb-F) reveals how protonation of the distal histidine (H64) changes the redox properties of the protein with a determined pKa of 5.3. In addition, fluoride binding in myoglobin provides a stabilization of -1.9 kcal/mol of the ferric Mb-F relative to ferric Mb without fluoride.

Conventional vs. micro-fat harvesting: how fat harvesting technique affects tissue-engineering approaches using adipose tissue-derived stem/stromal cells.

BACKGROUND: Biocompatible scaffolds as dermal substitutes are used commonly in soft tissue reconstruction and tissue-engineering approaches. The combination of these scaffolds with mesenchymal stem and stromal cells would have additional benefits in multilayer soft tissue reconstruction. In addition, the use of lipoaspirate may be beneficial for this purpose containing high levels of regenerative cells and relevant growth factors. However there are many factors, which may impact the lipoaspirate content of isolated cells, cell behaviour and growth factors. There is a lack of data as to whether fat-harvesting procedures using different cannulas of small diameter will impact these parameters, which are relevant not only for tissue engineering but also for clinical outcome. METHODS: Abdominal liposuctions were performed on 10 patients using the conventional fat harvesting by the Coleman cannula (3 mm, one-hole blunt tip) and the micro-fat-harvesting technique by the st'RIM cannula (2 mm, multi-perforated hole blunt tip) on contralateral area. Lipoaspirate contents of insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) were measured by enzyme-linked immunosorbent assay (ELISA). The in vitro viability of lipoaspirates was tested by the alamarBlue™ assay. Adipose-derived stem/stromal cells (ASCs) were isolated and the yields determined. Furthermore, ACSs were seeded on collagen elastin matrices (Matriderm™) and cell migration/adhesion rate was examined by the alamarBlue™ assay and visualised by two-photon microscopy. RESULTS: Conventionally obtained lipoaspirates were found to contain significantly higher concentrations of IGF and VEGF, but not PDGF or bFGF. No significant effects on the yields of ASCs or the in vitro viability of lipoaspirates obtained from different cannula sizes were observable. However, the viability and migration of isolated ASCs obtained from micro-harvested lipoaspirates were significantly higher. Moreover, a significant high adherence rate of isolated ASCs from the micro-fat-harvesting technique onto matrices was observed. CONCLUSION: The different sizes and surface/volume ratios of pieces of fatty tissue obtained by using different cannula sizes may be responsible for the observed differences and effects. Thus, micro-fat harvesting may be more suitable for tissue-engineering and -regenerative approaches using ASCs and collagen elastin matrices.

Cotransfection of vascular endothelial growth factor-A and platelet-derived growth factor-B via recombinant adeno-associated virus resolves chronic ischemic malperfusion role of vessel maturation.

OBJECTIVES: We set out to investigate the ability of cardiotropic adeno-associated viral vector (AAV2.9 = recombinant adeno-associated virus [rAAV]) to induce prolonged expression of vascular endothelial growth factor (VEGF)-A and platelet-derived growth factor (PDGF)-B in a rabbit hindlimb ischemia model and a pig model of hibernating myocardium. BACKGROUND: Gene therapy to induce angiogenesis and arteriogenesis has produced mixed results. However, long-acting viruses, such as rAAV, as well as combined induction of angiogenesis and vessel maturation might extend the therapeutic potential. METHODS: In rabbits, 0.5 x 10(11) particles rAAV.VEGF-A with or without 1 x 10(12) particles rAAV.PDGF-B were retroinfused at day 7 after femoral artery excision. At days 7 and 35, collateral counts and perfusion were determined, each value given as the day 35/day 7 ratio. Capillary-to-muscle fiber ratio was determined at day 35. In pigs, implantation of a reduction stent graft into the circumflex artery led to complete occlusion at day 28. At this time point, retroinfusion of rAAV.VEGF-A (1 x 10(13) particles), rAAV.VEGF-A/PDGF-B (2 x 10(12) and 4 x 10(12) particles, respectively) or mock transfection was performed. Ejection fraction and left ventricular end-diastolic pressure were assessed at days 28 and 56. RESULTS: In rabbits, rAAV.VEGF-A strongly induced angiogenesis (capillary-to-muscle fiber ratio; 1.67 +/- 0.09 vs. 1.32 +/- 0.11 in rAAV.LacZ-treated limbs, p < 0.05), but not collateral growth (125 +/- 7% vs. 106 +/- 7%, p = NS) or perfusion (136 +/- 12% vs. 107 +/- 9%, p = NS). With VEGF-A/PDGF-B cotransfection, collateral growth increased to 146 +/- 9%, perfusion to 163 +/- 8% of the respective day 7 value (p < 0.05). In the pig model, retroinfusion of rAAV.VEGF-A/PDGF-B increased regional myocardial blood flow reserve from 101 +/- 4% (rAAV.Mock) to 129 +/- 8% (p < 0.05), based on collateral growth (3.2 +/- 0.3 in rAAV.Mock vs. 9.0 +/- 0.4 in rAAV.VEGF-A/PDGF-B, p < 0.05), whereas rAAV.VEGF-A did not alter flow reserve (112 +/- 7%) or collateral count (5.2 +/- 0.7). rAAV.VEGF-A/PDGF-B improved ejection fraction (55 +/- 5% vs. 34 +/- 3% in rAAV.Mock, p < 0.05) unlike rAAV.VEGF-A (37 +/- 2%). CONCLUSIONS: Retroinfusion of rAAV.VEGF-A alone induces angiogenesis, but fails to enhance collateralization and perfusion, unless PDGF-B is cotransfected. In addition to neovascularization, rAAV.VEGF-A/PDGF-B improves regional and global myocardial function in hibernating myocardium.

Cytotoxin ClyA from Escherichia coli assembles to a 13-meric pore independent of its redox-state.

ClyA is a pore-forming toxin from virulent Escherichia coli and Salmonella enterica strains. Here, we show that the intrinsic hemolytic activity of ClyA is independent of its redox state, and that the assembly of both reduced and oxidized ClyA to the ring-shaped oligomer is triggered by contact with lipid or detergent. A rate-limiting conformational transition in membrane-bound ClyA monomers precedes their assembly to the functional pore. We obtained a three-dimensional model of the detergent-induced oligomeric complex at 12 A resolution by combining cryo- and negative stain electron microscopy with mass measurements by scanning transmission electron microscopy. The model reveals that 13 ClyA monomers assemble into a cylinder with a hydrophobic cap region, which may be critical for membrane insertion.

Mechanism of the electron transfer catalyst DsbB from Escherichia coli.

The membrane protein DsbB from Escherichia coli is essential for disulfide bond formation and catalyses the oxidation of the periplasmic dithiol oxidase DsbA by ubiquinone. DsbB contains two catalytic disulfide bonds, Cys41-Cys44 and Cys104-Cys130. We show that DsbB directly oxidizes one molar equivalent of DsbA in the absence of ubiquinone via disulfide exchange with the 104-130 disulfide bond, with a rate constant of 2.7 x 10 M(-1) x s(-1). This reaction occurs although the 104-130 disulfide is less oxidizing than the catalytic disulfide bond of DsbA (E(o)' = -186 and -122 mV, respectively). This is because the 41-44 disulfide, which is only accessible to ubiquinone but not to DsbA, is the most oxidizing disulfide bond in a protein described so far, with a redox potential of -69 mV. Rapid intramolecular disulfide exchange in partially reduced DsbB converts the enzyme into a state in which Cys41 and Cys44 are reduced and thus accessible for reoxidation by ubiquinone. This demonstrates that the high catalytic efficiency of DsbB results from the extreme intrinsic oxidative force of the enzyme.