BackgroundAge-related macular degeneration (AMD), a complex disease involving genetic variants and environmental insults, is among the leading causes of blindness in Western populations. Genetic and histologic evidence implicate the complement system in AMD pathogenesis; and smoking is the major environmental risk factor associated with increased disease risk. Although previous studies have demonstrated that cigarette smoke exposure (CE) causes retinal pigment epithelium (RPE) defects in mice, and smoking leads to complement activation in patients, it is unknown whether complement activation is causative in the development of CE pathology; and if so, which complement pathway is required.MethodsMice were exposed to cigarette smoke or clean, filtered air for 6 months. The effects of CE were analyzed in wildtype (WT) mice or mice without a functional complement alternative pathway (AP; CFB−/−) using molecular, histological, electrophysiological, and behavioral outcomes.ResultsCE in WT mice exhibited a significant reduction in function of both rods and cones as determined by electroretinography and contrast sensitivity measurements, concomitant with a thinning of the nuclear layers as measured by SD-OCT imaging and histology. Gene expression analyses suggested that alterations in both photoreceptors and RPE/choroid might contribute to the observed loss of function, and visualization of complement C3d deposition implies the RPE/Bruch's membrane (BrM) complex as the target of AP activity. RPE/BrM alterations include an increase in mitochondrial size concomitant with an apical shift in mitochondrial distribution within the RPE and a thickening of BrM. CFB−/− mice were protected from developing these CE-mediated alterations.ConclusionsTaken together, these findings provide clear evidence that ocular pathology generated in CE mice is dependent on complement activation and requires the AP. Identifying animal models with RPE/BrM damage and verifying which aspects of pathology are dependent upon complement activation is essential for developing novel complement-based treatment approaches for the treatment of AMD.

Background Brain death (BD) can immunologically prime the donor organ and is thought to lead to exacerbated ischemia reperfusion injury (IRI) post-transplantation. Using a newly developed mouse model of BD, we investigated the effect of donor BD on post transplant cardiac IRI. We further investigated the therapeutic effect of a targeted complement inhibitor in recipients of BD donor hearts, and addressed the clinical relevance of these studies by analysis of human heart biopsies from BD and domino (living) donors. Methods and Results Hearts from living or brain dead donor C57BL/6 mice were transplanted into C57BL/6 or BALB/c recipients. Recipient mice were treated with the complement inhibitor CR2-Crry or vehicle control (n=6). Isografts were analyzed 48 hours post-transplant for injury, inflammation and complement deposition, and allografts monitored for graft survival. Human cardiac biopsies were analyzed for complement deposition and inflammatory cell infiltration. In the murine model, donor BD exacerbated IRI and graft rejection as demonstrated by increased myocardial injury, serum cardiac troponin, cellular infiltration, inflammatory chemokine and cytokine levels, complement deposition, and decreased graft survival. CR2-Crry treatment of recipients significantly reduced all measured outcomes in grafts from both BD and living donors compared to controls. Analysis of human samples documented the relevance of our experimental findings and revealed exacerbated complement deposition and inflammation in grafts from BD donors compared to grafts from living donors. Conclusions BD exacerbates post-transplant cardiac IRI in mice and humans, and decreases survival of mouse allografts. Further, targeted complement inhibition in recipient mice ameliorates BD-exacerbated IRI.

PurposeMorphologic and genetic evidence exists that an overactive complement system driven by the complement alternative pathway (AP) is involved in pathogenesis of age-related macular degeneration (AMD). Smoking is the only modifiable risk factor for AMD. As we have shown that smoke-related ocular pathology can be prevented in mice that lack an essential activator of AP, we ask here whether this pathology can be reversed by increasing inhibition in AP.MethodsMice were exposed to either cigarette smoke (CS) or filtered air (6 hours/day, 5 days/week, 6 months). Smoke-exposed animals were then treated with the AP inhibitor (CR2-fH) or vehicle control (PBS) for the following 3 months. Spatial frequency and contrast sensitivity were assessed by optokinetic response paradigms at 6 and 9 months; additional readouts included assessment of retinal morphology by electron microscopy (EM) and gene expression analysis by quantitative RT-PCR.ResultsThe CS mice treated with CR2-fH showed significant improvement in contrast threshold compared to PBS-treated mice, whereas spatial frequency was unaffected by CS or pharmacologic intervention. Treatment with CR2-fH in CS animals reversed thinning of the retina observed in PBS-treated mice as analyzed by spectral-domain optical coherence tomography, and reversed most morphologic changes in RPE and Bruch's membrane seen in CS animals by EM.ConclusionsTaken together, these findings suggest that AP inhibitors not only prevent, but have the potential to accelerate the clearance of complement-mediated ocular injury. Improving our understanding of the regulation of the AP is paramount to developing novel treatment approaches for AMD.

The etiology of mitochondrial disease is poorly understood. Furthermore, treatment options are limited, and diagnostic methods often lack the sensitivity to detect disease in its early stages. Disrupted oxidative phosphorylation (OXPHOS) that inhibits ATP production is a common phenotype of mitochondrial disorders that can be induced in zebrafish by exposure to 2,4-dinitrophenol (DNP), a FDA-banned weight-loss agent and EPA-regulated environmental toxicant, traditionally used in research labs as an uncoupler of OXPHOS. Despite the DNP-induced OXPHOS inhibition we observed using in vivo respirometry, the development of the DNP-treated and control zebrafish were largely similar during the first half of embryogenesis. During this period, DNP-treated embryos induced gene expression of mitochondrial and nuclear genes that stimulated the production of new mitochondria and increased glycolysis to yield normal levels of ATP. DNP-treated embryos were incapable of sustaining this mitochondrial biogenic response past mid-embryogenesis, as shown by significantly lowered ATP production and ATP levels, decreased gene expression, and the onset of developmental defects. Examining neural tissues commonly affected by mitochondrial disease, we found that DNP exposure also inhibited motor neuron axon arbor outgrowth and the proper formation of the retina. We observed and quantified the molecular and physiological progression of mitochondrial dysfunction during development with this new model of OXPHOS dysfunction, which has great potential for use in diagnostics and therapies for mitochondrial disease.

Sexual dimorphisms in weaponry and aggression are common in species in which one sex (usually males) competes for access to mates or resources necessary for reproduction -sexually dimorphic weaponry and aggression, in other words, are frequently the result of intrasexual selection. In snapping shrimp, the major chela (snapping claw) can be a deadly weapon, and males of many species have larger chelae than females, a pattern readily interpreted as resulting from intrasexual selection. Thus, males might be expected to show more sex-specific aggression than females, and be more aggressive overall. We tested these predictions in two species of snapping shrimp in a territorial defense context. Neither of these predictions was supported: in both species, females, but not males, engaged in sex-specific aggression and females were more aggressive than males overall. These contrasting sexual dimorphisms -larger weaponry in males but higher aggression in females -highlight the importance of considering the function of weaponry and aggression in contexts other than direct competitions over mates. In addition, species differences in the degree of sexual dimorphism in chela size were due to differences in female, not male, chela size, and the species with greater sexual dimorphism in weaponry was significantly less aggressive overall; also, while paired and solitary males did not differ in residual chela size, for the species with greater sexual dimorphism, females carrying embryos had smaller residual chela sizes. These results suggest that understanding the sexual dimorphisms in weaponry and aggression in snapping shrimp requires understanding the relative costs and benefits of both in females as well as males.