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Background: Patients with non-ST-segment elevation acute coronary syndrome (NSTE-ACS) have varying degrees of salvageable myocardium at risk of irreversible injury. We hypothesized that a novel model of NSTE-ACS produces acute myocardial injury, measured by increased T2 cardiovascular magnetic resonance (CMR), without significant necrosis by late gadolinium enhancement (LGE)., Methods: In a canine model, partial coronary stenosis was created and electrodes placed on the epicardium. Myocardial T2, an indicator of at-risk myocardium, was measured pre- and post-tachycardic pacing., Results: Serum troponin-I (TnI) was not detectable in unoperated sham animals but averaged 1.97 ± 0.72 ng/mL in model animals. Coronary stenosis and pacing produced significantly higher T2 in the affected vs. the remote myocardium (53.2 ± 4.9 vs. 43.6 ± 2.8 ms, p < 0.01) with no evident injury by LGE. Microscopy revealed no significant irreversible cellular injury. Relative respiration rate (RRR) of affected vs. remote myocardial tissue was significantly lower in model vs. sham animals (0.72 ± 0.07 vs. 1.04 ± 0.07, p < 0.001). Lower RRR corresponded to higher final TnI levels (R(2) = 0.83, p = 0.004) and changes in CaMKIID and mitochondrial gene expression., Conclusions: A large animal NSTE-ACS model with mild TnI elevation and without ST elevation, similar to the human syndrome, demonstrates signs of acute myocardial injury by T2-CMR without significant irreversible damage. Reduced tissue respiration and associated adaptations of critical metabolic pathways correspond to increased myocardial injury by serum biomarkers in this model. T2-CMR as a biomarker of at-risk but salvageable myocardium warrants further consideration in preclinical and clinical studies of NSTE-ACS.

Background: New evidence links nicotine to the regulation of T cell-mediated inflammation via a 7 nicotinic cholinergic receptor activation, and chronic nicotine exposure (smoking) reduces the incidence of granulomatous diseases. We sought to determine whether nicotine treatment was well tolerated while effectively normalizing immune responses in patients with active pulmonary sarcoidosis., Methods: Consenting adults with symptomatic sarcoidosis (n 5 13) were randomly assigned to receive 12 weeks of nicotine treatment plus conventional therapy or conventional therapy alone. Obtained blood cells were evaluated for their responsiveness to selected Toll-like receptor (TLR) and nucleotide oligomerization domain-like receptor ligands and T cell surface marker expression before and after nicotine treatment. Asymptomatic patients (n 5 6) and disease-free subjects (n 5 6) served as comparative control subjects. Adverse events were monitored for the duration of the study., Results: Compared with the asymptomatic group, symptomatic patients had impaired peripheral responses to TLR2, TLR4, and TLR9 ligands (anergy) and reduced peripheral populations of CD4 1 FoxP3 1 regulatory T cells (Tregs). Nicotine treatment was associated with restoration of TLR2 and TLR9 responsiveness, and expansion of Tregs, including the CD4 1 CD25 2 FoxP3 1 phenotype. There were no serious adverse events or signs of nicotine dependency., Conclusions: Nicotine treatment in active pulmonary sarcoidosis was well tolerated and restored peripheral immune responsiveness to TLR2 and TLR9 agonists and expansion of FoxP3 1 Tregs, including a specific “preactivated” (CD25 2 ) phenotype. The immune phenotype of patients with symptomatic sarcoidosis treated with nicotine closely resembled that of asymptomatic patients, supporting the notion that nicotine treatment may be beneficial in this patient population.

The prevalence of sarcoidosis in the United States is unknown, with estimates ranging widely from 1 to 40 per 100,000. We sought to determine the prevalence of sarcoidosis in our health system compared to other rare lung diseases and to further establish if the prevalence was changing over time. We interrogated the electronic medical records of all patients treated in our health system from 1995 to 2010 (1.48 million patients) using the common ICD9 codes for sarcoidosis (135), lung cancer (162), and several other lung diseases characterized, like sarcoidosis, as "rare lung diseases". The patient demographic information (race, gender, age) was further analyzed to identify signature data patterns. The prevalence of sarcoidosis in our health system increased steadily from 164/100,000 in 1995 to 330/100,000 in 2010, and this trend could not be ascribed simply to changes in patient demographics or patient referral patterns. We further estimate that the prevalence of sarcoidosis exceeds 48 per 100,000 in Franklin County, Ohio, the demographic profile of which is nearly identical to that of the U.S. Sarcoidosis prevalence increased over time relative to lung cancer, a benchmark disease with stable disease prevalence, and exceeded that of other rare lung diseases. We postulate that the observed 2-fold increase in sarcoidosis disease prevalence in our health system is primarily related to improved detection and diagnostic approaches, and we conclude that the actual prevalence of sarcoidosis in central Ohio greatly exceeds current U.S. estimates., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Mitochondrial morphology and function are altered in intestinal epithelia during endotoxemia. However, it is unclear whether mitochondrial abnormalities occur in lung epithelial cells during acute sepsis or whether mitochondrial dysfunction corresponds with altered epithelial barrier function. Thus, we hypothesized that the intestinal epithelium is more susceptible to mitochondrial injury than the lung epithelium during acute sepsis and that mitochondrial dysfunction precedes impaired barrier function. Using a resuscitated feline model of Escherichia coli-induced sepsis, lung and ileal tissues were harvested after 6 h for histological and mitochondrial ultrastructural analyses in septic (n = 6) and time-matched controls (n = 6). Human lung epithelial cells (HLEC) and Caco-2 monolayers (n = 5) were exposed to 'cytomix' (TNFα: 40 ng/ml, IL-1β: 20 ng/ml, IFNγ: 10 ng/ml) for 24-72 h, and measurements of transepithelial electrical resistance (TER), epithelial permeability and mitochondrial membrane potential (ΔΨ) were taken. Lung epithelial morphology, mitochondrial ultrastructure and pulmonary gas exchange were unaltered in septic animals compared to matching controls. While histologically intact, ileal epithelia demonstrated marked mitochondrial ultrastructural damage during sepsis. Caco-2 monolayers treated with cytomix showed a significant decrease in mitochondrial ΔΨ within 24 h, which was associated with a progressive reduction in TER and increased epithelial permeability over the subsequent 48 h. In contrast, mitochondrial ΔΨ and epithelial barrier functions were preserved in HLEC following cytomix. These findings indicate that intestinal epithelium is more susceptible to mitochondrial damage and dysfunction than the lung epithelium in the context of sepsis. Early alterations in mitochondrial function portend subsequent epithelial barrier dysfunction., (© 2011 The Authors. International Journal of Experimental Pathology © 2011 International Journal of Experimental Pathology.)

Mitochondrial antigens released from damaged cells act as "danger signals" capable of promoting innate immune cell migration and activation via formyl peptide receptors (FPRs). Lung epithelial cells are equipped to migrate and mount innate immune responses in the context of acute lung injury. The goal of this study was to determine whether lung epithelial cells express FPRs, which are capable of responding to mitochondrial antigens to promote wound closure and inflammation. Using human Beas2B lung epithelial cells grown to confluency and subjected to linear scratch injury, it was found that mitochondrial antigens enhanced epithelial wound closure, and this phenomenon was inhibited by cyclosporin H, a selective inhibitor of FPR. Although mitochondrial antigens also promoted IL-8 release, this release was not FPR dependent and was unrelated to FPR-induced lung epithelial cell wound closure. The expression of functional FPR was confirmed in Beas2B and primary human tracheobronchial epithelial cells, particularly in lamellipodia at the leading edge of the closing wound. The expression of FPR was increased in response to TNF-α, LPS, scratch injury, and mitochondrial antigen treatment. Considered together, these data confirm that human lung epithelial cells express functional FPRs, which are capable of responding to endogenous mitochondrial danger signals, to promote wound closure.

Rationale: Little is known about the genetic regulation of granulomatous inflammation in sarcoidosis., Objectives: To determine if tissue gene array analysis would identify novel genes engaged in inflammation and lung remodeling in patients with sarcoidosis., Methods: Gene expression analysis was performed on tissues obtained from patients with sarcoidosis at the time of diagnosis (untreated) (n = 6) compared with normal lung tissue (n = 6). Expression of select genes was further confirmed in lung tissue from a second series of patients with sarcoidosis and disease-free control subjects (n = 11 per group) by semi-quantitative RT-PCR. Interactive gene networks were identified in patients with sarcoidosis using Ingenuity Pathway Analysis (Ingenuity Systems, Inc., Redwood, CA) software. The expression of proteins corresponding to selected overexpressed genes was determined using fluorokine multiplex analysis, and immunohistochemistry. Selected genes and proteins were then analyzed in bronchoalveolar lavage fluid in an independent series of patients with sarcoidosis (n = 36) and control subjects (n = 12)., Measurements and Main Results: A gene network engaged in Th1-type responses was most significantly overexpressed in the sarcoidosis lung tissues, including genes not previously reported in the context of sarcoidosis (e.g., IL-7). MMP-12 and ADAMDEC1 transcripts were most highly expressed (> 25-fold) in sarcoidosis lung tissues, corresponding with increased protein expression by immunohistochemistry. MMP-12 and ADAMDEC1 gene and protein expression were increased in bronchoalveolar lavage samples from patients with sarcoidosis, correlating with disease severity., Conclusions: Tissue gene expression analyses provide novel insights into the pathogenesis of pulmonary sarcoidosis. MMP-12 and ADAMDEC1 emerge as likely mediators of lung damage and/or remodeling and may serve as markers of disease activity.

Objective: Zinc deficiency is common among populations at high risk for sepsis mortality, including elderly, alcoholic, and hospitalized patients. Zinc deficiency causes exaggerated inflammatory responses to endotoxin but has not been evaluated during bacterial sepsis. We hypothesized that subacute zinc deficiency would amplify immune responses and oxidant stress during bacterial sepsis {lsqb;i.e., cecal ligation and puncture (CLP){rsqb; resulting in increased mortality and that acute nutritional repletion of zinc would be beneficial., Design: Prospective, randomized, controlled animal study., Setting: University medical center research laboratory., Subjects: Adult male C57BL/6 mice., Interventions: Ten-week-old, male, C57BL/6 mice were randomized into three dietary groups: 1) control diet, 2) zinc-deficient diet for 3 weeks, and 3) zinc-deficient diet for 3 weeks followed by oral zinc supplementation for 3 days (n = 35 per diet). Mice were then assigned to receive either CLP or sham operation (n = 15 each per diet). CLP and sham-operated treatment groups were further assigned to a 7-day survival study (n = 10 per treatment per diet) or were evaluated at 24 hours (n = 5 per treatment per diet) for signs of vital organ damage., Measurements and Main Results: Sepsis mortality was significantly increased with zinc deficiency (90% vs. 30% on control diet). Zinc-deficient animals subject to CLP had higher plasma cytokines, more severe organ injury, including increased oxidative tissue damage and cell death, particularly in the lungs and spleen. None of the sham-operated animals died or developed signs of organ damage. Zinc supplementation normalized the inflammatory response, greatly diminished tissue damage, and significantly reduced mortality., Conclusions: Subacute zinc deficiency significantly increases systemic inflammation, organ damage, and mortality in a murine polymicrobial sepsis model. Short-term zinc repletion provides significant, but incomplete protection despite normalization of inflammatory and organ damage indices.

Objective: Accumulating evidence indicates that mitochondrial function is impaired in vital organs during sepsis. In addition to oxidative phosphorylation, mitochondria participate in diverse cellular functions ranging from protein and lipid metabolism to programmed cell death. We analyzed liver mitochondrial protein expression patterns (i.e., proteomics) during acute endotoxemia to discover novel insights into mitochondrial responses to acute systemic inflammation., Design: A normotensive endotoxemia model was employed in which altered mitochondrial morphology occurs under conditions minimizing the potentially confounding effects of tissue hypoxia and acidosis., Setting: University medical research laboratory., Subjects: Random-source, adult, male cats., Interventions: Hemodynamic resuscitation and maintenance of acid-base balance and tissue oxygen availability were provided to preserve baseline homeostatic conditions. Treatment groups received isotonic saline vehicle (control; n = 5) or endotoxin (lipopolysaccharide, LPS, at 3.0 mg/kg intravenously; n = 5]. Liver samples were obtained 4 h posttreatment, and mitochondrial proteins were isolated and quantitatively compared using two-dimensional gel electrophoresis. Differentially expressed proteins (> 1.5-fold change relative to controls) were identified using mass spectrometry., Measurements and Results: Among over 500 protein spots that were separated 14 were differentially expressed in mitochondria of LPS-treated animals relative to matching controls. Spectrometric analyses demonstrated increased expression of urea cycle enzymes, heat shock protein (HSP) 60 and manganese superoxide dismutase, whereas expression of HSP70, F(1)-ATPase and key enzymes regulating lipid metabolism was reduced., Conclusions: Considering the known functions of each of the proteins exhibiting altered expression, it is likely that the observed changes in liver mitochondrial protein expression are reflective of significant changes in mitochondrial function in response to endotoxemia.

Rationale: Cyclosporin A (CsA) is known to preserve cardiac contractile function during endotoxemia, but the mechanism is unclear. Increased nitric oxide (NO) production and altered mitochondrial function are implicated as mechanisms contributing to sepsis-induced cardiac dysfunction, and CsA has the capacity to reduce NO production and inhibit mitochondrial dysfunction relating to the mitochondrial permeability transition (MPT)., Objectives: We hypothesized that CsA would protect against endotoxin-mediated cardiac contractile dysfunction by attenuating NO production and preserving mitochondrial function., Methods: Left ventricular function was measured continuously over 4 h in cats assigned as follows: control animals (n = 7); LPS alone (3 mg/kg, n = 8); and CsA (6 mg/kg, n = 7), a calcineurin inhibitor that blocks the MPT, or tacrolimus (FK506, 0.1 mg/kg, n = 7), a calcineurin inhibitor lacking MPT activity, followed in 30 min by LPS. Myocardial tissue was then analyzed for NO synthase-2 expression, tissue nitration, protein carbonylation, and mitochondrial morphology and function., Measurements and Main Results: LPS treatment resulted in impaired left ventricular contractility, altered mitochondrial morphology and function, and increased protein nitration. As hypothesized, CsA pretreatment normalized cardiac performance and mitochondrial respiration and reduced myocardial protein nitration. Unexpectedly, FK506 pretreatment had similar effects, normalizing both cardiac and mitochondrial parameters. However, CsA and FK506 pretreatments markedly increased protein carbonylation in the myocardium despite elevated manganese superoxide dismutase activity during endotoxemia., Conclusions: Our data indicate that calcineurin is a critical regulator of mitochondrial respiration, tissue nitration, protein carbonylation, and contractile function in the heart during acute endotoxemia.

Food Drug and Cosmetic Blue No. 1 dye (FD&C Blue No. 1) is commonly added to enteral nutrition formulations in order to facilitate the detection of gastric aspirate in tracheal secretions of critically ill patients. However, reports of systemic blue dye absorption and associated adverse outcomes are emerging. We report two cases of abnormal systemic absorption of FD&C Blue No. 1 in critically ill patients who subsequently died of refractory shock and metabolic acidosis. Risk factors and mechanisms of FD&C Blue No. 1 toxicity are discussed, and alternate approaches to gastric aspiration detection in critically ill patients are considered.

The apoptogenic protein cytochrome c can be quantitated by reverse-phase HPLC, but this method is not utilized by those who investigate mechanisms of cell death. Here, we extend the sensitivity of the method to exceed that available from immunogenic approaches and report specific procedures for applying the method to preparations of intact mitochondria, and to supernatants and pellets that arise from mitochondrial incubations. The detection limit corresponds to 0.6% of total cytochrome c found in 100 microg of rat liver mitochondrial protein, or to all of the cytochrome c that is expected in approximately 6000 hepatocytes. A single determination can be completed in 20 min, compared to a time scale of days for Western blotting methods, or hours for ELISA-based methods. The procedures are illustrated by experiments that determine the amount of cytochrome c released following the mitochondrial permeability transition as a function of medium ionic strength, and by long-term incubations of intact mitochondria in the presence and absence of an exogenous oxidizable substrate. Swelling and the release of adenylate kinase activity have been determined simultaneously to show how the data can be applied to evaluate the role of outer membrane disruption in mechanisms that release cytochrome c.