Stephen P. Kantrow

Induction of arginase isoforms in the lung during hyperoxia

L-Arginine can be metabolized by nitric oxide (NO) synthase (NOS) to produce NO or by arginase to produce urea and L-ornithine. In the liver, arginase (the AI isoform) is a key enzyme in the urea cycle. In extrahepatic organs including the lung, the function of arginase (the AII isoform) is less clear. Because we found that lung AII was upregulated during 100% O2 exposure in preliminary experiments, we sought to characterize expression of the arginase isoforms and inducible NOS and to assess the functions of arginase in hyperoxic lung injury. Male Sprague-Dawley rats were exposed to 100% O2 for 60 h. Protein expression of AI and AII and their cellular distribution were determined. The activities of arginase and NOS were also measured. Expression of arginase was correlated with that of ornithine decarboxylase, a biochemical marker for tissue repair, in a separate group of rats allowed to recover in room air for 48 h. We found by Western blot analyses that both AI and AII proteins were upregulated after 60 h of hyperoxic exposure (403 and 88% increases by densitometry, respectively) and, like ornithine decarboxylase, remained elevated during the recovery phase. Arginase activity increased by 37%. Immunostaining showed that increases in AI and AII were mainly in the peribronchial and perivascular connective tissues. NOS activity was unchanged and inducible NOS was not induced, but the level of nitrogen oxides in the lung decreased by 67%. Our study showed in vivo induction of arginase isoforms during hyperoxia. The strong expression of arginase in the connective tissues suggests that the function of pulmonary arginase may be linked to connective tissue elements, e.g., fibroblasts, during lung injury and recovery.l-Arginine can be metabolized by nitric oxide (NO) synthase (NOS) to produce NO or by arginase to produce urea andl-ornithine. In the liver, arginase (the AI isoform) is a key enzyme in the urea cycle. In extrahepatic organs including the lung, the function of arginase (the AII isoform) is less clear. Because we found that lung AII was upregulated during 100% O2exposure in preliminary experiments, we sought to characterize expression of the arginase isoforms and inducible NOS and to assess the functions of arginase in hyperoxic lung injury. Male Sprague-Dawley rats were exposed to 100% O2 for 60 h. Protein expression of AI and AII and their cellular distribution were determined. The activities of arginase and NOS were also measured. Expression of arginase was correlated with that of ornithine decarboxylase, a biochemical marker for tissue repair, in a separate group of rats allowed to recover in room air for 48 h. We found by Western blot analyses that both AI and AII proteins were upregulated after 60 h of hyperoxic exposure (403 and 88% increases by densitometry, respectively) and, like ornithine decarboxylase, remained elevated during the recovery phase. Arginase activity increased by 37%. Immunostaining showed that increases in AI and AII were mainly in the peribronchial and perivascular connective tissues. NOS activity was unchanged and inducible NOS was not induced, but the level of nitrogen oxides in the lung decreased by 67%. Our study showed in vivo induction of arginase isoforms during hyperoxia. The strong expression of arginase in the connective tissues suggests that the function of pulmonary arginase may be linked to connective tissue elements, e.g., fibroblasts, during lung injury and recovery.

Regulation of tumor necrosis factor cytotoxicity by calcineurin.

Cyclosporin (CsA) inhibits mitochondrial death signaling and opposes tumor necrosis factor (TNF)‐induced apoptosis in vitro. However, CsA is also a potent inhibitor of calcineurin, a phosphatase that may participate in cell death. Therefore, we tested the hypothesis that calcineurin regulates TNF cytotoxicity in rat hepatoma cells (FTO2B). TNF‐treated FTO2B cells appeared apoptotic by DNA fragmentation, nuclear condensation, annexin V binding, and caspase activation. We studied two calcineurin inhibitors, CsA and FK506, and found that each potently inhibited TNF cytotoxicity. Western blot demonstrated calcineurin in FTO2B homogenates. In a model of mitochondrial permeability transition (MPT), we found that CsA prevented MPT and cytochrome c release, while FK506 inhibited neither. In summary, we present evidence that calcineurin participates in an apoptotic death pathway activated by TNF. CsA may oppose programmed cell death by inhibiting calcineurin activity and/or inhibiting mitochondrial signaling.

Stable Transgene Expression in Tumors and Metastases After Transduction with Lentiviral Vectors Based on Human Immunodeficiency Virus Type 1

The relatively low efficiency of target cell transduction and variations in the stability of transgene expression by retroviral vectors based on the Moloney murine leukemia virus (MoMLV) are major impediments to the use of such vectors in cancer gene therapy approaches. The present study was designed to investigate the stability and efficiency of transgene expression in human lung and breast cancer cell lines transduced with vectors based on human immunodeficiency virus type 1 (HIV-1) in vitro and in vivo in nude mouse models of metastasis. H460 lung carcinoma cells and MDA-MB-231 breast carcinoma cells were transduced with lentiviral vectors encoding enhanced green fluorescent protein (EGFP) and beta-galactosidase (beta-Gal), respectively. Transduced H460 cells were administered to nude mice by either intravenous or subcutaneous injection and MDA-MB-231 cells were implanted orthotopically into the mammary fat pad of such mice to induce primary tumor and metastatic lung tumor formation. High-level EGFP expression was maintained in transduced H460 cells in metastatic lung nodules for up to 6 weeks and transgene expression in vitro persisted for at least 23 days after retrieval of EGFP-positive H460 cells from the lungs of tumor-bearing mice and subsequent cultivation in vitro. Likewise, beta-Gal expression levels in metastatic MDA-MB-231 cells in lungs remained high for up to 11 weeks. Southern blot analyses carried out with DNA from lung nodules showed that proviral DNAs in H460 cells were maintained stably over many cell generations and during subsequent reimplantation in vivo. However, molecular analyses revealed variations in transgene copy numbers and expression levels among individual lung clones. These results demonstrate the usefulness of HIV-1-based lentiviral vectors for sustained and stable transgene expression in human lung and breast cancer cell lines in vitro and in vivo.

Proinflammatory cytokines increase in sepsis after anti-adhesion molecule therapy.

Cytokine mediators and leukocyte-endothelial cell adhesion molecules are critical and interdependent components of the acute inflammatory response in sepsis. We hypothesized that the administration of monoclonal antibodies to intercellular adhesion molecule-1 (CD54) or E- and L-selectin (CD62E/L) would decrease serum levels of the proinflammatory cytokines interleukin-1beta (IL-1), IL-6, and IL-8 and tumor necrosis factor receptor (TNFR-1) in baboons during sepsis. Adult male baboons received infusions of 1 x 10(9) colony forming units (CFU)/kg heat-killed Escherichia coli (E. coli) followed 12 h later by live E. coli (1 x 10(10) CFU/kg). At the time of live bacterial infusion, six septic animals were treated with a monoclonal antibody to CD54 and six with an antibody to CD62E and L (1 mg/kg). Eight untreated septic animals served as controls. Sequentially drawn serum samples were assayed for IL-1, IL-6, IL-8, and TNFR-1 using enzyme-linked immunoassay (ELISA). Data were compared using Mann-Whitney U tests and Chi-square analyses. Median survival was decreased in both treatment groups compared to controls (P < 0.05). Peak IL-1 level was higher than controls in septic animals treated with anti-CD54 but not anti-CD62E/L (P < 0.05, P = NS, respectively). Elevations in IL-6, IL-8, and TNFR-1 were increased and prolonged in both antibody treated groups compared to controls (P < 0.05). These results provide the first in vivo evidence that leukocyte-endothelial adhesion molecules CD54 and CD62E/L regulate cytokine production in sepsis.

Pulmonary Vasculitis: Clinical Presentation, Differential Diagnosis, and Management

This review focuses on vasculitides with prominent pulmonary manifestations and discusses key contributions from the recent literature. Pulmonary vasculitis should be considered when clinical findings include alveolar hemorrhage, nodular and cavitary lung disease, airway stenosis, pulmonary artery aneurysms, or pulmonary artery stenosis. The differential diagnostic considerations for common clinical presentations of vasculitis in the lung are important, and several recent additions are discussed. Treatment for established pulmonary vasculitis is effective and has decreased the morbidity and mortality associated with these diseases while introducing an increased risk of infectious complications. Advances in immunosuppressive therapy have improved treatment of refractory disease and are likely to change initial treatment strategies in the future.

Low-voltage electricity-induced lung injury: Electricity-induced lung injury

We report a case of bilateral pulmonary infiltrates and haemoptysis following low‐voltage electricity exposure in an agricultural worker. A 58‐year‐old man standing in water reached for an electric watering machine and sustained an exposure to 220 V circuit for an uncertain duration. The electricity was turned off by another worker, and the patient was asymptomatic for the next 10 h until he developed haemoptysis. A chest radiograph demonstrated bilateral infiltrates, and chest computed tomography (CT) revealed ground‐glass opacities with interstitial thickening. Evaluations, including electrocardiogram, serum troponin, N‐terminal pro‐B‐type natriuretic peptide (NT‐pro BNP), coagulation studies, and echocardiogram, found no abnormality. The patient was treated for suspected electricity‐induced lung injury and bleeding with tranexamic acid and for rhabdomyolysis with volume resuscitation. He recovered with complete resolution of chest radiograph abnormalities by Day 7. This is the first reported case of bilateral lung oedema and/or injury after electricity exposure without cardiac arrest.

A Letter From New Orleans Five Years Later

It has already been 5 years since Hurricane Katrina, the most costly natural disaster in American history, drowned New Orleans and the surrounding Gulf Coast. Moment by moment as levees softened and failed and flood waters poured in, scores of television cameras beamed indelible images of horrific human suffering into every living room across America. Over 1,800 people died as a direct result of Katrina, most of them homebound elderly in New Orleans. In addition, over 250,000 persons were rendered homeless and over half of those have been permanently displaced. No other event in American history, including the great flood of 1926, has displaced so many people. A full year after Hurricane Katrina, New Orleans’ population was less than 50% of what it had been before the storm. In one year New Orleans went from America’s 33rd largest city to its 89th. Although the events that unfolded in the late summer of 2005 had been predicted for over 50 years, the healthcare system of New Orleans, the State of Louisiana, and the U.S. Federal Government were each ill-prepared to deal with the magnitude of humanitarian need that resulted. Critical resources were either inadequate or poorly positioned to care for the 100,000 persons who either chose not to leave or could not leave in the days preceding the storm. The result was that thousands of mostly poor New Orleanians were stranded for days without food, water, shelter, or medical care. Even those trapped inside of New Orleans’ dark and silent hospitals, many with life-threatening medical problems, remained invisible to the outside world, including the incident command. Without any hope of outside help, patient survival had been squarely placed on the shoulders of tired, frightened, and often inexperienced healthcare workers. Somewhat surprisingly, it seemed that these same healthcare workers, who themselves suffered tremendous losses, turned out to be better suited for the task of nurturing and rescuing patients than the armed militias dispatched by the federal government. Such a struggle of an ICU team to save its critically ill patients inside of Charity Hospital was shared with this same readership 5 years ago (1). In the weeks following Katrina we received countless letters from well wishers expressing their condolences for what we had experienced. Our simple response at that time was to reassure them that the singular emotion that so many of us felt was gratitude for having had the opportunity to serve. For all of those trapped inside hospitals without the technological distractions of modern medicine, there were frequent unanticipated discoveries about the goodness of human beings. Amid all of the chaos, time seemed to slow down and acts of kindness by young resident physicians, nurses, and allied health professionals became commonplace. In the early months after the water was pumped out and the television cameras had been turned off, the realities of rebuilding began to set in. With three quarters of New Orleans housing uninhabitable, there was a mass exodus of anyone who could afford to leave and who had a place to go. The poor, who seem to wear homing beacons for disasters, found themselves left behind, housed in toxic mobile trailers in neighborhoods that lacked public transportation, schools, grocery stores, recreation facilities, places of worship, and importantly mental health facilities. At a time when outpatient clinics were nonexistent, only 3 of New Orleans’ 16 acute care hospitals were able to re-open, and 70% of physician practices were dislocated. Predictably, rates of crime, depression, and suicide doubled. If New Orleans had been on life support before Katrina, in the first year following Katrina, she had become ‘‘a DNR.’’ But the old and wise will remind us that disruptions on any scale, whether they affect whole communities or individuals, can offer us a gift: the opportunity to change. And change we did. Many institutions that had been crumbling before Katrina have been built anew. New Orleans’ public schools, which had been on a path to extinguish the light of learning for generations to come, have attracted the attention and service of educators and advocates of social justice from all over the world. And for many of us, it has turned out to be a blessing to be offered the opportunity to resurrect from the destruction the things we treasured, and to rebuild them from fragments, to choose all over again our work, our homes, our partners, our friends. Through evolution rather than revolution, day-to-day life has slowly become richer. Men and women of conscience from all over the country have descended on New Orleans with fresh ideas and boundless energy. Families have been reunited, new charter schools have opened, businesses have returned, and an NFL franchise has inspired a nation with the idea that with the right work ethic and perseverance, all things are possible. One brick at a time, the healthcare infrastructure in New Orleans is also being rebuilt, but now with a different focus. Community clinics and medical homes have replaced hospitals as the focal points for the delivery of services, and disease management programs have reduced hospitalizations for chronic medical conditions. Charity Hospital, one of the oldest hospitals in continuous operation in the United States and the major teaching hospital for Tulane and LSU Schools of Medicine, the place where ‘‘the unusual occurs and miracles happen’’ (Figure 1), remains closed and will likely never reopen again as a hospital. In its place a new

Chapter 8 Cellular injury in sepsis

2.4 billion state-of-the-art healthcare complex is being planned, with anticipated ground breaking just around the corner. This medical corridor will become the home of a new regional VA hospital and a new state-funded academic medical center scheduled to open in 2014. There is no doubt that the world is plagued with problems more devastating than Katrina. Poverty, hunger, disease, natural and human-caused disasters, and violence occur close to home and far away. The legacy of Hurricane Katrina will not be the blown down trees and the flooded homes; rather, it will be how this natural disaster yanked at a nation’s heart strings and

Release of cytochrome c from liver mitochondria during permeability transition.

Publisher Summary This chapter discusses the cellular injuries and responses that occur in sepsis. The mediators of cellular injury in sepsis are diverse and include endotoxin and other products derived from invading organisms and cytokines released by the host in response to endotoxin or other microbial components. These proinflammatory responses are so impressive that therapeutic strategies to prevent organ failure and death due to sepsis have been based on circumventing them. Some of these strategies have included antibodies directed against endotoxin and against circulating cytokines released in response to endotoxin—for example, tumor necrosis factor (TNF) and intedeukin-1 (IL-1). Soluble receptors to TNF and IL-1 have also been infused during sepsis in an attempt to prevent their binding to host cells. Multiple lines of investigation suggest that endotoxin and cytokines are capable of directly injuring host cells, and some of the possible mechanisms include inhibition of cellular respiration, oxidative injury and induction of nitric oxide synthesis. The most serious early hemodynamic response to severe sepsis is hypotension. The etiology of this hypotension is multifactorial. Systemic vasodilation and increased capillary permeability occur after release of potent vasodilators and injury to the vascular endothelium. Prominent among these vasodilators is nitric oxide (NO·), produced by a calcium independent, inducible NO· synthase (iNOS) expressed in endothelium, smooth muscle and a variety of other cells throughout the body. The endothelium and vascular smooth muscle are both sites of production and targets for its vasodilator activity.