Enhancer selection dictates gene expression responses in remote organs during tissue regeneration.

Acute trauma stimulates local repair mechanisms but can also impact structures distant from the injury, for example through the activity of circulating factors. To study the responses of remote tissues during tissue regeneration, we profiled transcriptomes of zebrafish brains after experimental cardiac damage. We found that the transcription factor gene cebpd was upregulated remotely in brain ependymal cells as well as kidney tubular cells, in addition to its local induction in epicardial cells. cebpd mutations altered both local and distant cardiac injury responses, altering the cycling of epicardial cells as well as exchange between distant fluid compartments. Genome-wide profiling and transgenesis identified a hormone-responsive enhancer near cebpd that exists in a permissive state, enabling rapid gene expression in heart, brain and kidney after cardiac injury. Deletion of this sequence selectively abolished cebpd induction in remote tissues and disrupted fluid regulation after injury, without affecting its local cardiac expression response. Our findings suggest a model to broaden gene function during regeneration in which enhancer regulatory elements define short- and long-range expression responses to injury.

A Genetic Cardiomyocyte Ablation Model for the Study of Heart Regeneration in Zebrafish.

Adult zebrafish possess an elevated cardiac regenerative capacity as compared with adult mammals. In the past two decades, zebrafish have provided a key model system for studying the cellular and molecular mechanisms of innate heart regeneration. The ease of genetic manipulation in zebrafish has enabled the establishment of a genetic ablation injury model in which over 60% of cardiomyocytes can be depleted, eliciting signs of heart failure. After this severe injury, adult zebrafish efficiently regenerate lost cardiomyocytes and reverse heart failure. In this chapter, we describe the methods for inducing genetic cardiomyocyte ablation in adult zebrafish, assessing cardiomyocyte proliferation, and histologically analyzing regeneration after injury.

Impact of Surveillance After Lobectomy for Lung Cancer on Disease Detection and Survival.

INTRODUCTION: Existing guidelines for surveillance after non-small-cell lung cancer (NSCLC) treatment are inconsistent and have relatively sparse supporting literature. This study characterizes detection rates of metachronous and recurrent disease during surveillance with computed tomography scans after definitive treatment of early stage NSCLC. MATERIALS AND METHODS: The incidence of metachronous and recurrent disease in patients who previously underwent complete resection via lobectomy for stage IA NSCLC at a single center from 1996 to 2010 were evaluated. A subgroup analysis was used to compare survival of patients whose initial surveillance scan was 6 ± 3 months (early) versus 12 ± 3 months (late) after lobectomy. RESULTS: Of 294 eligible patients, 49 (17%) developed recurrent disease (14 local only, 35 distant), and 45 (15%) developed new NSCLC. Recurrent disease was found at a mean of 22 ± 19 months, and new primaries were found at a mean of 52 ± 31 months after lobectomy (P < .01). Five-year survival after diagnosis of recurrent disease was significantly lower than after diagnosis of second primaries (2.3% vs. 57.5%; P < .001). In the subgroup analysis of 187 patients, both disease detection on the initial scan (2% [2/94] vs. 4% [4/93]; P = .44) and 5-year survival (early, 80.8% vs. late, 86.7%; P = .61) were not significantly different between the early (n = 94) and the late (n = 93) groups. CONCLUSION: Surveillance after lobectomy for stage IA NSCLC is useful for identifying both new primary as well as recurrent disease, but waiting to start surveillance until 12 ± 3 months after surgery is unlikely to miss clinically important findings.

Serologic reactivity to the emerging pathogen Granulibacter bethesdensis.

BACKGROUND: Granulibacter bethesdensis is a recently described member of the Acetobacteraceae family that has been isolated from patients with chronic granulomatous disease (CGD). Its pathogenesis, environmental reservoir(s), and incidence of infection among CGD patients and the general population are unknown. METHODS: Detected antigens were identified by mass spectroscopy after 2-dimensional electrophoresis and immunoaffinity chromatography. The prevalence of Granulibacter immunoreactivity was assessed through immunoblotting and enzyme-linked immunosorbent assay (ELISA). RESULTS: Methanol dehydrogenase (MDH) and formaldehyde-activating enzyme were recognized during analysis of sera from infected patients. Unique patterns of immunoreactive bands were identified in Granulibacter extracts, compared with extracts of other Acetobacteraceae species. By use of criteria based on these specific bands, specimens from 79 of 175 CGD patients (45.1%) and 23 of 93 healthy donors (24.7%) reacted to all 11 bands. An ELISA that used native MDH to capture and detect immunoglobulin G was developed and revealed high-titer MDH seroreactivity in culture-confirmed cases and 5 additional CGD patients. Testing of samples collected prior to culture-confirmed infection demonstrated instances of recent seroconversion, as well as sustained seropositivity. Infection of CGD mice with G. bethesdensis confirmed acquisition of high-titer antibody-recognizing MDH. CONCLUSIONS: These serologic tests suggest that Granulibacter immunoreactivity is more common among CGD patients and, perhaps, among healthy donors than was previously suspected. This finding raises the possibility that clinical presentations of Granulibacter infection may be underappreciated.

Innate immunity against Granulibacter bethesdensis, an emerging gram-negative bacterial pathogen.

Acetic acid bacteria were previously considered nonpathogenic in humans. However, over the past decade, five genera of Acetobacteraceae have been isolated from patients with inborn or iatrogenic immunodeficiencies. Here, we describe the first studies of the interactions of the human innate immune system with a member of this bacterial family, Granulibacter bethesdensis, an emerging pathogen in patients with chronic granulomatous disease (CGD). Efficient phagocytosis of G. bethesdensis by normal and CGD polymorphonuclear leukocytes (CGD PMN) required heat-labile serum components (e.g., C3), and binding of C3 and C9 to G. bethesdensis was detected by immunoblotting. However, this organism survived in human serum concentrations of ≥90%, indicating a high degree of serum resistance. Consistent with the clinical host tropism of G. bethesdensis, CGD PMN were unable to kill this organism, while normal PMN, in the presence of serum, reduced the number of CFU by about 50% after a 24-h coculture. This finding, together with the observations that G. bethesdensis was sensitive to H(2)O(2) but resistant to LL-37, a human cationic antimicrobial peptide, suggests an inherent resistance to O(2)-independent killing. Interestingly, 10 to 100 times greater numbers of G. bethesdensis were required to achieve the same level of reactive oxygen species (ROS) production induced by Escherichia coli in normal PMN. In addition to the relative inability of the organism to elicit production of PMN ROS, G. bethesdensis inhibited both constitutive and FAS-induced PMN apoptosis. These properties of reduced PMN activation and resistance to nonoxidative killing mechanisms likely play an important role in G. bethesdensis pathogenesis.

Recurrent Granulibacter bethesdensis infections and chronic granulomatous disease.

Chronic granulomatous disease (CGD) is characterized by frequent infections, most of which are curable. Granulibacter bethesdensis is an emerging pathogen in patients with CGD that causes fever and necrotizing lymphadenitis. However, unlike typical CGD organisms, this organism can cause relapse after clinical quiescence. To better define whether infections were newly acquired or recrudesced, we use comparative bacterial genomic hybridization to characterize 11 isolates obtained from 5 patients with CGD from North and Central America. Genomic typing showed that 3 patients had recurrent infection months to years after apparent clinical cure. Two patients were infected with the same strain as previously isolated, and 1 was infected with a genetically distinct strain. This organism is multidrug resistant, and therapy required surgery and combination antimicrobial drugs, including long-term ceftriaxone. G. bethesdensis causes necrotizing lymphadenitis in CGD, which may recur or relapse.