A novel immune competent murine hypertrophic scar contracture model: A tool to elucidate disease mechanism and develop new therapies

Hypertrophic scar (HSc) contraction following burn injury causes contractures. Contractures are painful and disfiguring. Current therapies are marginally effective. To study pathogenesis and develop new therapies, a murine model is needed. We have created a validated immune‐competent murine HSc model. A third‐degree burn was created on dorsum of C57BL/6 mice. Three days postburn, tissue was excised and grafted with ear skin. Graft contraction was analyzed and tissue harvested on different time points. Outcomes were compared with human condition to validate the model. To confirm graft survival, green fluorescent protein (GFP) mice were used, and histologic analysis was performed to differentiate between ear and back skin. Role of panniculus carnosus in contraction was analyzed. Cellularity was assessed with 4′,6‐diamidino‐2‐phenylindole. Collagen maturation was assessed with Picro‐sirius red. Mast cells were stained with Toluidine blue. Macrophages were detected with F4/80 immune. Vascularity was assessed with CD31 immune. RNA for contractile proteins was detected by quantitative real‐time polymerase chain reaction (qRT‐PCR). Elastic moduli of skin and scar tissue were analyzed using a microstrain analyzer. Grafts contracted to ～45% of their original size by day 14 and maintained their size. Grafting of GFP mouse skin onto wild‐type mice, and analysis of dermal thickness and hair follicle density, confirmed graft survival. Interestingly, hair follicles disappeared after grafting and regenerated in ear skin configuration by day 30. Radiological analysis revealed that panniculus carnosus doesn't contribute to contraction. Microscopic analyses showed that grafts show increase in cellularity. Granulation tissue formed after day 3. Collagen analysis revealed increases in collagen maturation over time. CD31 stain revealed increased vascularity. Macrophages and mast cells were increased. qRT‐PCR showed up‐regulation of transforming growth factor beta, alpha smooth muscle actin, and rho‐associated protein kinase 2 in HSc. Tensile testing revealed that human skin and scar tissues are tougher than mouse skin and scar tissues.     

Comparison of plasma N-brain natriuretic peptide, peak oxygen consumption, and left ventricular ejection fraction for severity of chronic heart failure

We investigated whether a simple blood test of plasma N-brain natriuretic peptide (N-BNP), compared with echocardiographic left ventricular ejection fraction (LVEF), both measured at rest, correlated well with aerobic exercise capacity (peak oxygen consumption [VO₂]) in patients with chronic heart failure. Plasma N-BNP was found to significantly correlate with peak VO₂ (p <0.001) and exercise duration (p = 0.001), whereas LVEF showed very poor correlations with peak VO₂ and exercise duration (both p >0.3). The results suggest that N-BNP actually reflects functional cardiac impairment better than LVEF.     

Zileuton,a 5-lipoxygenase inhibitor,increases production of thromboxane A2 and platelet aggregation in patients with asthma

Leukotrienes, generated from arachidonic acid via the lipoxygenase pathway, play an important role in the pathophysiology of asthma. Therefore, leukotriene inhibitors, such as Zileuton, are used in the treatment of asthma. However, thromboxanes, generated from arachidonic acid via the cyclooxygenase pathway, play an important role in platelet aggregation and thrombosis. Therefore, we studied whether Zileuton, by shifting arachidonic acid to the cyclooxygenase pathway, enhances thromboxane production and, hence, platelet aggregation. Blood samples were collected from 10 asthmatic patients before and 2 weeks after standard Zileuton treatment. Spontaneous platelet aggregation was measured in platelet-rich plasma. Platelet-rich plasma was also used to determine thromboxane B(2), a stable metabolite of thromboxane A(2), as the indirect measure of thromboxane A(2) because thromboxane A(2) is too unstable for assay. Baseline thromboxane B(2) and platelet aggregation values in the 10 asthmatic patients were normal. Treatment with Zileuton for 2 weeks significantly increased thromboxane B(2) levels from baseline levels of 267 +/- 54 microg/l to 389 +/- 62 microg/l after 2 weeks of treatment (P < 0.0002). Spontaneous platelet aggregation also increased significantly from baseline values of 4.2 +/- 2.4% to 6.8 +/- 2.8% after 2 weeks of treatment (P < 0.0001). These results establish that Zileuton, an effective drug for asthma, adversely affects in vitro platelet function. The findings suggest that this drug, and perhaps related agents also, may pose a thrombotic risk; clinical attention will be needed to address this possibility.     

Functional role of cysteine-146 in Escherichia coli thymidylate synthase.

Analysis of mutant Escherichia coli thymidylate synthases (EC 2.1.1.45) with various amino acids substituted for cysteine at position 146 revealed the cysteine to be involved in the binding of 2'-deoxyuridylate as well as initiating the catalytic process. The substitution of a serine or alanine residue at position 146 did not appreciably alter the binding affinity for 2'-deoxyuridylate but the serine mutant enzyme was less active by a factor of 5000, whereas the alanine mutant enzyme was catalytically inactive. In contrast, the substitution of a glycine or threonine at position 146 created inactive enzymes with higher 2'-deoxyuridylate dissociation constants. The dissociation constant values for 2'-deoxyuridylate were used to estimate the overall contribution of the side chain of the amino acid at position 146 to substrate binding. The results suggested that the side chains of cysteine, alanine, and serine make nonspecific but effective van der Waals contacts with 2'-deoxyuridylate, thereby contributing about 0.82 kcal.mol-1 (1 cal = 4.184 J) to the apparent binding energy of the substrate.     

Cyclophosphamide type I hypersensitivity in systemic lupus erythematosus

Cyclophosphamide is an important immunosuppressive agent in the treatment of many rheumatic diseases. Urticaria and anaphylaxis to intravenous cyclophosphamide (i.v. CYC) have been reported in patients with haematological and solid organ malignancies. This is the first report in the rheumatology literature of a type I hypersensitivity reaction following monthly i.v. CYC. An 18-year-old girl with systemic lupus erythematosus (SLE) developed generalized urticaria (without concomitant angioedema or anaphylaxis) following i.v. CYC. She had previously developed life-threatening angioedema following a respiratory tract infection. She successfully completed regular pulse i.v. CYC with pre-medication with anti-histamine. In the absence of a severe type I hypersensitivity reaction and other suitable immunosuppressive agents, i.v. CYC may be safely continued with pre-medication and careful monitoring during each infusion.     

Recruitment of individually (all-or-none) responding cells, rather than amplitude enhancement, is the single-cell mechanism subserving the dose-responsive activation of intracellular calcium second messenger signaling by the human lutinizing-hormone receptor

We have investigated at the single-cell level how the human LH receptor mediates a dose-responsive increase in intracellular free calcium-ion concentrations ([Ca²⁺]_i). In human embryonic kidney cells (293 cells) stably transfected with the full-length human LH receptor cDNA. Intact dimeric LH, but not LH β- or α-subunits, evoked specific [Ca²⁺]_i signals. High-resolution fluorescence (fura-2) video-microscopy demonstrated cell-to-cell variability in [Ca²⁺]_i signaling responses in individual cells, viz., an all-or-none spike (9%), spike-and-plateau (25%), or plateau (52%) types of temporal signal. Oscillatory [Ca²⁺]_i responses were observed in 12–14% of LH-stimulated cells unrelated to LH concentration. The LH dose-response originated by higher concentrations of LH recruiting more individually responding cells (rather than altering [Ca²⁺]_i signal amplitude), and eliciting a [Ca²⁺]_i rise more rapidly, i.e., at reduced latency. Cobalt did not abolish the LH-stimulated [Ca²⁺]_i spike-and-lateau response, but decreased the percentage of cells with a plateau pattern. Quench experiments demonstrated influx of Mn²⁺ following the [Ca²⁺]_i spike, thus directly documenting divalent cation inflow during the plateau phase. Adenylyl-cyclase activation with forskolin or treatment with a cAMP analog failed to elicit the biphasic [Ca²⁺]_i resoonse, and pertussis toxin (PTX) did not alter LH-stimulated [Ca²⁺]_i signaling. However, overnight preincubation with LH reduced the percentage of [Ca²⁺]_i-responding cells following re-exposure to LH to 5.7% (vs 72% in control), suggesting LH-induced desensitization of the LH-receptor directed [Ca²⁺]_i signal. In summary, the present studies of human LH receptor signal transduction at the single-cell level show that increasing concentrations of LH achieve a dose-dependent intracellular Ca²⁺ signaling response by recruiting an increasing number of [Ca²⁺]_i-responding cells, while concomitantly decreasing the temporal latency of the biphasic [Ca²⁺]_i signal without altering the amplitude of its spike phase. Prolonged exposure to LH appears to desensitize the LH receptor-driven [Ca²⁺]_i signal.     

Plasma platelet activating factor degradation and serum lipids after coronary bypass surgery.

OBJECTIVE: Platelet activating factor (PAF) is a potent mediator in inflammatory responses and maybe involved in various disease states. Degradation of PAF in plasma results from the action of a specific, lipoprotein associated, acetylhydrolase. The aim was to determine plasma acetylhydrolase activity under optimised conditions, PAF half life, phospholipase A2 activity, the lyso-derivative of PAF (lyso-PAF), and lipids in patients undergoing coronary artery bypass grafting. METHODS: The study variables were determined 3 d and 7 d following coronary artery surgery and compared to presurgical values in 15 males, age 55(SEM 4) years. RESULTS: Three days following coronary bypass grafting, total, LDL and HDL cholesterol fell significantly by 30%, 45%, and 15% respectively (p less than 0.001), all decreases correlating with bypass time (p less than 0.025). Concentrations remained low at 7 d (p less than 0.005). Acetylhydrolase activity fell by 38% (p less than 0.001) at 3 d post-surgery and remained depressed, but plasma PAF half life did not change after surgery. The inverse relationship between acetylhydrolase activity and plasma PAF half life preoperatively (p less than 0.01) was not evident after surgery. There was a direct linear relationship between acetylhydrolase activity and both total (p less than 0.002) and LDL cholesterol (p less than 0.001) before surgery. The fall in acetylhydrolase activity correlated with the fall in these lipids (p less than 0.01) but not with that of HDL cholesterol. Plasma lyso-PAF decreased by 65% (p less than 0.001) at 3 d and remained depressed (p less than 0.001). Plasma phospholipase A2 activity increased by 60% (p less than 0.01) and remained raised (p less than 0.05), the increase at 3 d being related to bypass time (p less than 0.05). CONCLUSIONS: The large fall in plasma acetylhydrolase activity after coronary bypass grafting is consistent with the fall in plasma lipids. However, the absence of a significant change in the measured PAF half life in plasma raises questions as to the pathophysiological significance of the decrease in acetylhydrolase activity.