PBDE-47 and PBDE mixture (DE-71) toxicities and liver transcriptomic changes at PND 22 after in utero/postnatal exposure in the rat.

Pentabromodiphenyl ethers (PBDE) are found in human tissue, in household dust, and in the environment, and a particular concern is the potential for the induction of cancer pathways from these fat-soluble persistent organic pollutants. Only one PBDE cancer study has been conducted and that was for a PBDE mixture (DE-71). Because it is not feasible to test all PBDE congeners in the environment for cancer potential, it is important to develop a set of biological endpoints that can be used in short-term toxicity studies to predict disease outcome after long-term exposures. In this study, PBDE-47 was selected as the test PBDE congener to evaluate and compare toxicity to that of the carcinogenic PBDE mixture. The toxicities of PBDE-47 and the PBDE mixture were evaluated at PND 22 in Wistar Han rat (Crl: WI (Han)) pups after in utero/postnatal exposure (0, 0.1, 15, or 50 mg/kg; dams, GD6-21; pups, PND 12-PND 21; oral gavage daily dosing). By PND 22, PBDE-47 caused centrilobular hypertrophy and fatty change in liver, and reduced serum thyroxin (T4) levels; similar effects were also observed after PBDE mixture exposure. Transcriptomic changes in the liver included induction of cytochrome p450 transcripts and up-regulation of Nrf2 antioxidant pathway transcripts and ABC membrane transport transcripts. Decreases in other transport transcripts (ABCG5 & 8) provided a plausible mechanism for lipid accumulation, characterized by a treatment-related liver fatty change after PBDE-47 and PBDE mixture exposure. The benchmark dose calculation based on liver transcriptomic data was generally lower for PBDE-47 than for the PBDE mixture. The up-regulation of the Nrf2 antioxidant pathway and changes in metabolic transcripts after PBDE-47 and PBDE mixture exposure suggest that PBDE-47, like the PBDE mixture (NTP 2016, TR 589), could be a liver toxin/carcinogen after long-term exposure.

A structured strategy to combine education for advanced MIS training in surgical oncology training programs.

Changing realities in surgery and surgical technique have heightened the need for agile adaptation in training programs. Current guidelines reflect the growing acceptance and adoption of the use of minimally invasive surgery (MIS) in oncology. North American general surgery residents are often not adequately skilled in advanced laparoscopic surgery skills at the completion of their residency. Presently, advanced laparoscopic surgery training during surgical oncology fellowship training occurs on an ad-hoc basis in many surgical oncology programs. We present a rational and template for a structured training in advanced minimally invasive surgical techniques during surgical oncology fellowship training. The structure of the program seeks to incorporate evidence-based strategies in MIS training from a comprehensive review of the literature, while maintaining essential elements of rigorous surgical oncology training. Fellows in this stream will train and certify in the Fundamentals of Laparoscopic Surgery (FLS) course. Fellows will participate in the didactic oncology seminar series continuously throughout the 27 months training period. Fellows will complete one full year of dedicated MIS training, followed by 15 months of surgical oncology training. Minimal standards for case volume will be expected for MIS cases and training will be tailored to meet the career goals of the fellows. We propose that a formalized MIS-Surgical Oncology Fellowship will allow trainees to benefit from an effective training curriculum and furthermore, that will allow for graduates to lead in a cancer surgery milieu increasingly focused on minimally invasive approaches.

Applications of physiologically based pharmacokinetic (PBPK) modeling and simulation during regulatory review.

Physiologically based pharmacokinetic (PBPK) modeling and simulation is a tool that can help predict the pharmacokinetics of drugs in humans and evaluate the effects of intrinsic (e.g., organ dysfunction, age, genetics) and extrinsic (e.g., drug-drug interactions) factors, alone or in combinations, on drug exposure. The use of this tool is increasing at all stages of the drug development process. This report reviews recent instances of the use of PBPK in decision-making during regulatory review. The examples are based on Center for Drug Evaluation and Research reviews of several submissions for investigational new drugs (INDs) and new drug applications (NDAs) received between July 2008 and June 2010. The use of PBPK modeling and simulation facilitated the following types of decisions: the need to conduct specific clinical pharmacology studies, specific study designs, and appropriate labeling language. The report also discusses the challenges encountered when PBPK modeling and simulation were used in these cases and recommends approaches to facilitating full utilization of this tool.

Metabolic dysfunction and depletion of mitochondria in hearts of septic rats.

Our previous studies indicate that hearts from septic rats have decreased work with oxygen wasting. The present studies test if there is energy deficit, changes in cardiac mitochondrial content and caspase activation during sepsis. Anesthetized, male Sprague-Dawley rats received no surgical treatment (control), laparotomy (sham), or laparotomy with cecal ligation and puncture (CLP) to induce polymicrobial septic shock. Hearts were isolated 12-14 h later. Cardiac work, oxygen consumption, substrate oxidation and energy stores were measured in perfused hearts. Normalized density of mitochondria was determined in ventricles without perfusion by morphometric analysis with electron microscopy. Citrate synthase activity was assessed in homogenates and isolated mitochondria. Cardiac work decreased significantly in CLP (47%), while oxygen consumption and glucose oxidation were unchanged compared with control or sham hearts (oxygen and substrate wasting). Tissue adenosine triphosphate, creatine phosphate and glycogen were lower in CLP hearts (energy deficit). Mitochondrial grid intersects decreased significantly from 151 +/- 8 sham to 130 +/- 4 CLP out of 361 possible intersects and autophagy was observed in CLP hearts. Total activity of citrate synthase decreased in homogenates (99 +/- 8 micromol/min/g wet weight sham vs. 62 +/- 7 CLP, P < 0.05) and in the mitochondrial fraction (27 +/- 1 micromol/min/g wet weight sham to 22 +/- 1 CLP, P < 0.05). Calculated mitochondrial content decreased from 63 +/- 4 mg protein/g wet weight sham to 46 +/- 5 CLP, P < 0.05 (mitochondrial depletion). Caspase-3 activity doubled and tumor necrosis factor alpha content tripled in CLP hearts. CONCLUSIONS. - Oxygen and substrate wasting in CLP occurs with fewer mitochondria and energy deficit, processes that are coincident with caspase-3 activation.

The role of endogenous NADPH oxidases in airway and pulmonary vascular smooth muscle function.

Reactive oxygen species generated from NADPH oxidase(s) in airway smooth muscle cells and pulmonary artery smooth muscle cells are important signaling intermediates. Nox4 appears to be the predominant gp91 homologue in these cells. However, expression of NADPH oxidase components is dependent on phenotype, and different homologues may be expressed during different functional states of the cell. NADPH oxidase(s) appear to be important not only for mitogenesis by these cells, but also for O(2) sensing. The regulation of NADPH oxidase(s) in airway and pulmonary artery smooth muscle cells has important implications for the pathobiochemistry of asthma and pulmonary vascular diseases.

Redox signaling of NF-kappaB by membrane NAD(P)H oxidases in normal and malignant cells.

Evidence is rapidly accumulating that low-activity NAD(P)H oxidases homologous to that in phagocytic cells generate reactive oxygen species as signaling intermediates. In this review we discuss evidence that signaling NAD(P)H oxidases in part influence normal and malignant cell division by activating the redox-regulated transcription factor nuclear factor kappaB. The roles of growth-regulatory NAD(P)H oxidases in human airway smooth muscle and malignant melanoma are used as examples.

Reactive oxygen species from NAD(P)H:quinone oxidoreductase constitutively activate NF-kappaB in malignant melanoma cells.

The transcription factor nuclear factor-kappaB (NF-kappaB) is constitutively activated in malignancies from enhanced activity of inhibitor of NF-kappaB (IkappaB) kinase, with accelerated IkappaBalpha degradation. We studied whether redox signaling might stimulate these events. Cultured melanoma cells generated superoxide anions (O(2)(-)) without serum stimulation. O(2)(-) generation was reduced by the NAD(P)H:quinone oxidoreductase (NQO) inhibitor dicumarol and the quinone analog capsaicin, suggesting that electron transfer from NQO through a quinone-mediated pathway may be an important source of endogenous reactive oxygen species (ROS) in tumor cells. Treatment of malignant melanoma cells with the H(2)O(2) scavenger catalase, the sulfhydryl donor N-acetylcysteine, the glutathione peroxidase mimetic ebselen, or dicumarol decreased NF-kappaB activation. Catalase, N-acetylcysteine, ebselen, dicumarol, and capsaicin also inhibited growth of melanoma and other malignant cell lines. These results raise the possibility that ROS produced endogenously by mechanisms involving NQO can constitutively activate NF-kappaB in an autocrine fashion and suggest the potential for new antioxidant strategies for interruption of oxidant signaling of melanoma cell growth.

Nonanticoagulant heparin inhibits NF-kappaB activation and attenuates myocardial reperfusion injury.

Heparin reduces ischemia-reperfusion injury to myocardium. This effect has been attributed to complement inhibition, but heparin also has other activities that might diminish ischemia-reperfusion. To further probe these mechanisms, we compared heparin or an o-desulfated nonanticoagulant heparin with greatly reduced anticomplement activity. When given at the time of coronary artery reperfusion in a canine model of myocardial infarction, heparin or o-desulfated heparin equally reduced neutrophil adherence to ischemic-reperfused coronary artery endothelium, influx of neutrophils into ischemic-reperfused myocardium, myocardial necrosis, and release of creatine kinase into plasma. Heparin or o-desulfated heparin also prevented dysfunction of endothelial-dependent coronary relaxation following ischemic injury. In addition, heparin and o-desulfated heparin inhibited translocation of the transcription nuclear factor-kappaB (NF-kappaB) from the cytoplasm to the nucleus in human endothelial cells and decreased NF-kappaB DNA binding in human endothelium and ischemic-reperfused rat myocardium. Thus heparin and nonanticoagulant heparin decrease ischemia-reperfusion injury by disrupting multiple levels of the inflammatory cascade, including the novel observation that heparins inhibit activation of the proinflammatory transcription factor NF-kappaB.

In vitro and in vivo responses to interleukin 12 are maintained until the late SIV infection stage but lost during AIDS.

The in vitro proliferative responses of macaque peripheral blood mononuclear cells (PBMCs) to IL-12 appeared similar before and early after SIV infection, whereas macaque PBMCs sampled during symptomatic stages of SIV infection showed markedly decreased responses. IL-12 was administered to SIVmac239-infected rhesus macaques either during the asymptomatic or the AIDS stage of infection in efforts to evaluate the effect of this cytokine on immune responses, viral loads, and hematopoietic functions in vivo. IFN-gamma secretion levels induced during the asymptomatic or early symptomatic phase were similar to preinfection induced levels, whereas in later AIDS stages this response was lost. The constitutive levels of other measured cytokines were not affected by IL-12 administration in vivo. The frequency and activity of circulating NK cells were markedly enhanced at early stages but not at symptomatic stages of SIV infection. pCTL frequencies were enhanced at early symptomatic stages but not at late AIDS stages. Despite its immunomodulatory effect, IL-12 did not seem to exacerbate or inhibit the replication of SIV in vivo, or the frequency of circulating infected lymphocytes. IL-12 administration was associated with a significant yet subclinical and transient decrease in hematocrit and hemoglobin levels without evidence of hemolysis, hemodilution, or reduction in the frequency of colony-forming unit potential of bone marrow CD34+ cells. This phenomenon may be explained by a functional inhibition of differentiation rather than an altered generation of bone marrow precursors. Thus, these results suggest that IL-12 may benefit HIV-1-infected patients only as long as their immune system retains its capability to respond to cytokine stimulation.

Requirement for reactive oxygen species in serum-induced and platelet-derived growth factor-induced growth of airway smooth muscle.

Reactive oxygen species have been recently identified as important mediators of mitogenic signaling in a number of cell types. We therefore explored their role in mediating mitogenesis of airway smooth muscle. The antioxidants catalase, N-acetylcysteine, and probucol significantly reduced proliferation in primary cultures of rat tracheal smooth muscle stimulated with fetal bovine serum or platelet-derived growth factor, without affecting cell viability or inducing apoptosis. N-Acetylcysteine also significantly reduced serum-stimulated elevation of c-Fos but did not prevent the normal mitogen-induced increase in c-fos mRNA. Fractionation of ribosomes by sucrose density centrifugation and subsequent dot-blot Northern analysis revealed that antioxidants reduced incorporation of c-fos mRNA into the heaviest polyribosomes, suggesting redox regulation of c-fos mRNA translation. Serum treatment of monolayers produced a small but reproducibly significant rise in superoxide dismutase-inhibitable reduction of ferricytochrome c by myocyte monolayers. Serum-induced ferricytochrome c reduction, cellular proliferation, and c-Fos elevation were decreased by the flavoprotein-dependent enzyme inhibitor dipheyleneiodonium. Growth responses to fetal bovine serum and superoxide dismutase-inhibitable reduction of ferricytochrome c were not different between cultured tracheal myocytes from wild-type versus gp91 phagocyte oxidase null mice. These results suggest that mitogen stimulation of airway smooth muscle induces signal transduction of cell proliferation that is in part dependent on generation of partially reduced oxygen species, generated by an NADH or NADPH oxidoreductase that is different from the oxidase in phagocytic cells.

Markedly elevated levels of interferon (IFN)-gamma, IFN-alpha, interleukin (IL)-2, IL-10, and tumor necrosis factor-alpha associated with fatal Ebola virus infection.

The role of immune mechanisms in the pathogenesis of Ebola hemorrhagic fever (EHF) remains to be elucidated. In this report, the serum cytokine levels of patients who died of EHF were compared with those of patients who recovered and those of control patients. A marked elevation of interferon (IFN)-gamma levels (>100 pg/mL) was observed in sequential serum samples from all fatal EHF cases compared with patients who recovered or controls. Markedly elevated serum levels of interleukin (IL)-2, IL-10, tumor necrosis factor (TNF)-alpha, and IFN-alpha were also noted in fatal EHF cases; however, they had a greater degree of variability. No differences were noted in serum levels of IL-4 and IL-6. mRNA quantitation from blood clots of the same patients showed relatively elevated levels of TNF-alpha and IFN-alpha in samples from EHF patients. Taken together, these results suggest that a high degree of immune activation accompanies and potentially contributes to a fatal outcome in EHF patients.

Molecular cloning and expression of rhesus macaque and sooty mangabey interleukin 16: biologic activity and effect on simian immunodeficiency virus infection and/or replication.

Interleukin 16 (IL-16) has been shown to diminish HIV and SIV replication through inhibition of HIV and SIV mRNA transcription. To evaluate its role further, we compared IL-16 cloned from disease-susceptible rhesus macaques and disease-resistant sooty mangabeys. Recombinant rhesus macaque (rr) IL-16 was compared with recombinant sooty mangabey (rm), human, and other nonhuman primate IL-16 sequences and evaluated for its ability to induce chemotaxis and inhibit the mixed lymphocyte response (MLR). Also, rrIL-16 and rmIL-16 were evaluated for suppression of SIVmac251, which replicates efficiently in T cells and monocyte/macrophages (dual tropic), and cloned SIVmac239, which replicates efficiently in T cells (T tropic). Sequence comparison of rrIL-16 and rmIL-16 with human IL-16 showed >97% amino acid identity. Biocharacterization of rrIL-16 revealed potent induction of chemotaxis (p < 0.05) and marked inhibition of MLR (73 +/- 0.6%,p < 0.05) in rhesus and human cell systems. Using rrIL-16 and rmIL-16, p27 antigen production from PBMCs infected with SIVmac251 was decreased up to 70% (p < 0.05 and p < 0.01, respectively). In similar cultures infected with SIVmac239, rrIL-16 and rmIL-16 reduced p27 levels by 96 and 100%, respectively. These data demonstrate the biologic and antiviral functionality of rrIL-16 and rmIL-16.

Expression and in vitro evaluation of rhesus macaque wild type (wt) and modified CC chemokines.

Several human CC chemokines have been shown to inhibit HIV/ SIV infection in vitro, providing the rationale for their potential use in vivo. However, because of their inherent physiological effect, such chemokines are reasoned to be of limited therapeutic value due to potential side effects. The knowledge that amino terminus modified or deleted human RANTES retains its receptor binding properties but loses its signaling properties has provided a means to use such modified chemokines in vivo for possible therapeutic benefits. In efforts to test the efficacy of such modified chemokines, our laboratory has cloned, sequenced, and prepared recombinant forms of wild-type (wt) and amino-terminus modified rhesus macaque chemokines MIP-1alpha, MIP-1beta, and RANTES. These sets of chemokines were tested for their potential to inhibit SIV infection and induce signaling. The data showed that whereas wt chemokines retained both virus inhibitory and signaling functions, corresponding amino-terminus modified chemokines only showed virus inhibitory effects without detectable signaling effects. Such reagents will be valuable for evaluation of their therapeutic potential in vivo, either alone or as adjuncts to other chemotherapeutic drugs.

Immune and hematopoietic parameters in HIV-1-infected chimpanzees during clinical progression toward AIDS.

Until recently, chimpanzees were considered susceptible to human immunodeficiency virus type 1 (HIV-1) infection, but refractory to disease induction based on the asymptomatic status of all experimentally infected chimpanzees after over 10 years postinfection (PI). However, a decline in peripheral CD4+ T cells was noted in one chimpanzee (C499) of the Yerkes cohort of HIV-1 infected apes, after 11 years PI concurrent with increasing plasma viral load. These clinical signs were followed by the occurrence of opportunistic infections, thrombocytopenia, and progressive anemia leading to euthanasia. A second chimpanzee (C455) was transfused with blood from C499 collected during the symptomatic stage. Shortly thereafter, this second animal showed a rapid decline in peripheral CD4+ T-cell levels and sustained high viral load. Hematological analyses showed a 50% decrease in CFU-GM for both apes during the symptomatic phase and a reduction of 40% and 73% of the total CFU despite normal levels of CD34+ cells in the bone marrow. Cryopreserved sequential PBMC samples from these two chimpanzees were analyzed for constitutive and PHA-P induced levels of cytokines and chemokines. Data show that whereas there were no detectable constitutive levels of mRNA coding for IL-2, 4, and 10, there appears to be a transient increase in IFN-gamma message level coincident with increased viremia and this IFN-gamma synthesis decreased with disease progression. PHA-induced cytokine mRNA analysis showed low or undetectable levels of IL-4 and IL-10 mRNA in all samples and a marked decrease in the levels of IL-2 shortly after HIV infection. In addition, there was also a gradual decrease in IFN-gamma mRNA with progression of disease. Of interest were the findings of high to normal levels of PHA-induced synthesis of the chemokines MIP-1alpha, MIP-1beta, and RANTES in samples during the asymptomatic and early symptomatic period, which also dramatically decreased at late stages of the disease. These data suggest important roles for IL-2, IFN-gamma, and the chemokines in the regulation of immune responses in HIV-1-infected chimpanzees.

Comparative sequence analysis of cytokine genes from human and nonhuman primates.

Two major issues severely limit the studies of human recombinant cytokines/growth factors in nonhuman primates. First, assays and reagents specific for the detection and quantitation of human cytokines do not all function when utilized to detect/quantitate the nonhuman primate cytokines. Second, although most of the human cytokines appear to induce similar, if not identical, biologic function when used with cells from nonhuman primates in vitro or in vivo, they invariably induce Ab responses in vivo, precluding their repeated and/or continued use in vivo. Our laboratory has thus initiated studies to clone, sequence, and prepare recombinant cytokines from nonhuman primates and to define assays and reagents for their detection and quantitation at the nucleic acid and protein level. The data that were derived from such studies show that the nonhuman primate cytokines IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 alpha, IL-12 beta, IL-15, IFN-alpha, IFN-gamma, and TNF-alpha share 93 to 99% homology at the nucleic acid and protein level with the human equivalents. The most prominent differences between human and nonhuman primate cytokine sequences were noted for IL-1 alpha/beta, IL-2, IL-8, IFN-alpha, IFN-gamma, and IL-12 beta. The aligned sequences of cytokines for human and several nonhuman primate species are provided herein, and a phylogenetic analysis of the published sequences of select cytokines from other species, along with those of the nonhuman primates, are described. In addition, comparative analysis of the relative bioactivity of our immunoaffinity-purified recombinant rhesus macaque IL-4, IL-15, and IFN-gamma with commercially available human recombinant cytokines is described herein.

Epitope mapping of the branched chain alpha-ketoacid dehydrogenase dihydrolipoyl transacylase (BCKD-E2) protein that reacts with sera from patients with idiopathic dilated cardiomyopathy.

Sera from 29 of 48 patients with idiopathic dilated cardiomyopathy (IDCM) and six of six patients with dilated cardiomyopathy (DCM) secondary to suspected viral myocarditis were shown to react with the branched chain alpha-ketoacid dehydrogenase (BCKD) complex mitochondrial proteins. Whereas sera from only 1 of 26 patients with ischemic heart disease showed reactivity against the BCKD complex protein, 0 of 30 sera from normal human volunteers, 0 of 64 sera from patients with lupus, and 0 of 34 sera from patients with rheumatoid arthritis showed detectable reactivity, denoting an element of specificity for the reactivity of sera from IDCM patients. The major reactivity was localized to the dihydrolipoyl transacylase (E2) component of BCKD complex. By using recombinant techniques, the immunodominant BCKD-E2 epitope recognized by sera from IDCM patients was localized to amino acid (aa) sequences 116 to 134. Each of the IDCM sera that reacted with the native BCKD complex was shown to react with the immunodominant peptide, as defined by a peptide inhibition ELISA and by an ELISA using the reactive peptide conjugated to BSA. Sera from IDCM patients that reacted with the native BCKD complex and the reactive peptide also showed inhibition of BCKD enzyme activity. The possible mechanisms for the induction of the Abs and the implications of these findings for the pathogenesis of IDCM are discussed.

Central venous catheter infections in pediatric patients--in a community hospital.

We reviewed the records of 23 pediatric patients who had received at least one central venous catheter during a two-year period. Nine patients had acute lymphoblastic leukemia (ALL), nine had other hematologic/oncologic diagnoses, and five had cystic fibrosis. Twenty-nine of 65 febrile episodes in 16 patients were associated with a catheter-related infection. Twenty of 40 catheters were associated with an infection over a period of 7,229 catheter days. For every 1,000 catheter days, four episodes of infections were observed. The number of infections/1,000 catheter days, the average life of a catheter (approximately equal to 180 days), and mean number of days elapsing before the first infection were not significantly different in the three diagnostic groups. Broviac catheters were used most often (24/40), followed by Quinton (9/40) and Port-a-Cath (7/40). Broviac catheters lasted twice as long (224 days, p less than 0.01) as Quinton and Port-a-Cath. Gram-positive cocci were isolated most frequently and Staphylococcus epidermidis was the most common pathogen. No consistent relationship between an absolute neutrophil count of less than 1,000/mm3 and infection with gram-positive cocci was seen. However, seven of eight episodes of gram-negative bacillary infections occurred in patients with an absolute neutrophil count of less than 1,000/m3 (p less than 0.005). Those patients who were not considered terminally ill responded well to antimicrobials. Catheter removal was necessary in only two instances.

Instrumental analysis of trace elements in thumbnails of human subjects.

Human thumbnails were analyzed for trace elements by instrumental analysis using thermal neutron activation technique. The average concentration of metals studied in clinically symptom-free adult female and male subjects were: zinc, 184 vs. 153 ppm; chromium, 6.8 vs. 4.2; selenium, 0.9 vs. 0.6; gold, 2.6 vs. 0.4; mercury, 1.9 vs. 0.4; silver, 0.7 vs. 0.3; cobalt, 0.07 vs. 0.04. A summary of literature reported concentration of metals in human nail is also presented.