Angiotensin II stimulation of NAD(P)H oxidase activity: upstream mediators

Angiotensin II (Ang II)-stimulated hypertrophy of vascular smooth muscle cells is mediated by reactive oxygen species (ROS) derived from NAD(P)H oxidases. The upstream signaling mechanisms by which Ang II activates these oxidases are unclear but may include protein kinase C, tyrosine kinases, phosphatidylinositol-3-kinase, and Rac, a small molecular weight G protein. We found that Ang II-stimulated ROS production is biphasic. The first phase occurs rapidly (peak at 30 seconds) and is dependent on protein kinase C activation. The larger second phase of ROS generation (peak at 30 minutes) requires Rac activation, because inhibition of Rac by either Clostridium difficile toxin A or dominant-negative Rac significantly inhibits Ang II-induced ROS production. Phosphatidylinositol-3-kinase inhibitors (wortmannin or LY294002) and the epidermal growth factor (EGF) receptor kinase blocker AG1478 attenuate both Rac activation and ROS generation. The upstream activator of EGF receptor transactivation, c-Src, is also required for ROS generation, because PP1, an Src kinase inhibitor, abrogates the Ang II stimulation of both responses. These results suggest that c-Src, EGF receptor transactivation, phosphatidylinositol-3-kinase, and Rac play important roles in the sustained Ang II-mediated activation of vascular smooth muscle cell NAD(P)H oxidases and provide insight into the integrated signaling mechanisms whereby Ang II stimulation leads to activation of the growth-related NAD(P)H oxidases.     

Functionally distinct dendritic cell (DC) populations induced by physiologic stimuli: prostaglandin E(2) regulates the migratory capacity of specific DC subsets

Migration of antigen (Ag)-loaded dendritic cells (DCs) from sites of infection into draining lymphoid tissues is fundamental to the priming of T-cell immune responses. We evaluated monocyte-derived DCs (MoDCs) and peripheral blood DCs (PBDCs) to respond to proinflammatory mediators, CD40L, and intact bacteria. All classes of stimuli induced DC phenotypic maturation. However, for MoDCs, only prostaglandin E(2) (PGE(2))-containing stimuli induced migratory-type DCs. Thus, immature MoDCs that encountered proinflammatory cytokines or CD40L or intact bacteria in the presence of PGE(2) acquired migratory capacity but secreted low levels of cytokines. Conversely, MoDCs that encountered pathogens or CD40L alone become nonmigratory cytokine-secreting cells (proinflammatory type). Interestingly, both migratory- and proinflammatory-type DCs expressed equivalent levels of chemokine receptors, suggesting that the role of PGE(2) was to switch on migratory function. We demonstrate that PGE(2) induces migration via the E-prostanoid 2/E-prostanoid 4 (EP(2)/EP(4)) receptors and the cAMP pathway. Finally, migratory-type MoDCs stimulated T-cell proliferation and predominantly IL-2 secretion, whereas proinflammatory-type MoDCs induced IFN-gamma production. In contrast, CD1b/c(+) PBDC rapidly acquired migratory capacity irrespective of the class of stimulus encountered and secreted low levels of cytokines. This suggests that not all mature stages of DCs are destined to migrate to lymphoid organs and that the sequence in which stimuli are encountered significantly affects which functions are expressed. Thus, certain immature DC subsets recruited from the resting precursor pool may have multiple functional fates that play distinct roles during the induction and effector phases of the immune response. These findings have important implications for the clinical utility of DCs in immunotherapy.     

Subclinical impairment of brain function in chronic hepatitis C infection

BACKGROUND/AIMS: Central nervous system abnormalities such as fatigue and depression occur more frequently in chronic hepatitis C virus (HCV) infection than in many other causes of chronic liver disease. The finding that fatigue is unrelated to activity of hepatitis or mode of infection could indicate an independent effect of HCV on brain function. This study tested the hypothesis of a subclinical cognitive dysfunction in HCV-infected patients. METHODS: One-hundred untreated HCV-RNA positive biopsy-proven patients were investigated by P300 event-related potentials, a sensitive electrophysiologic test of cognitive processing. Health-related quality of life and fatigue were assessed using the SF-36 questionnaire and the Fatigue Impact Scale, respectively. RESULTS: Cognitive brain function was subclinically impaired in the cohort of HCV-infected patients as indicated by significantly prolonged P300 latencies (P=0.01 for comparison to matched healthy subjects) and reduced P300 amplitudes (P<0.001, respectively). Seventeen of the 100 HCV-infected patients had P300 latencies outside the age-adjusted normal range. Abnormal P300 characteristics were not related to the degree of histologic or biochemical activity of hepatitis, severity of fatigue or mental health impairment. CONCLUSIONS: This study demonstrates that patients with HCV infection showed a slight but significant neurocognitive impairment, possibly indicating a further extrahepatic manifestation of chronic hepatitis C.     

Case Reports on Emergency Treatment of Cardiovascular Syndromes Through Heparin‐Mediated Low‐Density Lipoprotein/Fibrinogen Precipitation: A New Approach to Augment Cerebral and Myocardial Salvage

Abstract: We report the first experiences with HELP apheresis as an emergency treatment for acute cardiovascular syndromes; two patients who were not eligible for lysis therapy and catheter intervention were treated with HELP apheresis instead. Both patients had a most severe, generalized atherosclerosis and reached the hospital too late for conventional measures. In both cases, the use of the apheresis dramatically improved the clinical situation to such an extent that the possibilities of this apheresis system urge further investigation.     

Modular degradable dendrimers enable small RNAs to extend survival in an aggressive liver cancer model

RNA-based cancer therapies are hindered by the lack of delivery vehicles that avoid cancer-induced organ dysfunction, which exacerbates carrier toxicity. We address this issue by reporting modular degradable dendrimers that achieve the required combination of high potency to tumors and low hepatotoxicity to provide a pronounced survival benefit in an aggressive genetic cancer model. More than 1,500 dendrimers were synthesized using sequential, orthogonal reactions where ester degradability was systematically integrated with chemically diversified cores, peripheries, and generations. A lead dendrimer, 5A2-SC8, provided a broad therapeutic window: identified as potent [EC₅₀ < 0.02 mg/kg siRNA against FVII (siFVII)] in dose–response experiments, and well tolerated in separate toxicity studies in chronically ill mice bearing MYC-driven tumors (>75 mg/kg dendrimer repeated dosing). Delivery of let-7g microRNA (miRNA) mimic inhibited tumor growth and dramatically extended survival. Efficacy stemmed from a combination of a small RNA with the dendrimer’s own negligible toxicity, therefore illuminating an underappreciated complication in treating cancer with RNA-based drugs.Since the discovery of RNAi and the recognition of its therapeutic potential, there has been a continuous search for optimal delivery carriers (1–10). Tremendous progress has been made with regard to delivery efficacy of small RNAs to healthy livers, but the clinically required combination of high delivery potency to tumors and low hepatotoxicity is not currently met by existing delivery vehicles. This challenge represents an unappreciated complication in treating cancer with RNA-based drugs. Primary liver cancer, a chronic consequence of liver cirrhosis, is a leading cause of cancer death and a major global health problem (11). Unfortunately, all five phase III human clinical trials of small-molecule drugs for hepatocellular carcinoma (HCC) treatment failed within the past four years in part because debilitating, late-stage liver dysfunction amplifies drug toxicity (11, 12). MicroRNAs (miRNAs) present a promising alternative cancer treatment strategy because they can function as tumor suppressors by concurrently targeting multiple pathways involved in cell differentiation, proliferation, and survival (13–19). In this study, we used a highly aggressive, inducible MYC-driven transgenic liver cancer model where the chance for successful therapy is very limited, in equal part due to the rapidly growing cancer and the compromised function of the overtaken liver. Tumors grow endogenously from within the liver in this genetically engineered model, which is biologically faithful and lethal, in contrast to standard xenograft or orthotopic models derived from cell line implantation (20). The development of dendrimer carriers that can mediate a therapeutic benefit in this difficult-to-treat model was set as the paramount goal of the study. Notably, we find that high potency and low toxicity are of critical importance in the context of aggressive liver cancer models, where the carrier’s own toxicity can negate the benefit of on-target small-RNA therapies.To achieve this balance of low toxicity and high potency, we reasoned that one must more precisely examine the influence of chemical structure by expanding the structural diversity and molecular size of delivery carriers. Dendrimers represented an ideal system for this goal because they possess the same high degree of molecular uniformity as small molecules and the broad theoretical space for chemical tuning as polydisperse polymers (21–23). These intrinsic characteristics enable dendrimers to have unique properties for various biomedical applications (24–26). In gene delivery, most studies have used the limited number of commercial dendrimers for further chemical modification (27–29). However, orthogonal click chemistry has recently made a transformative impact on the synthesis of dendritic materials through protecting group-free methodologies to overcome historical limitations in fidelity and provide a larger toolbox for dendrimer design (30–34). The expansion of dendrimer applications therefore depends on the ability to easily tune the size, chemistry, topology, and, ultimately, dendrimer physical properties through chemical synthesis.We hypothesized that, through introduction of ester bonds and molecular diversity at each expansion step, modular degradable dendrimers would possess a critical balance of low toxicity and high delivery potency (Fig. 1). We further speculated that chemical modulation of cores, peripheries, and generations may be accelerated by utilization of efficient click reactions to enable a large increase in both the total number (>1,500) and chemical diversity of dendrimers. Biocompatible, degradable esters were included as a key design component because all RNAi therapies are transient and require sustained and repeated treatment. This ultimately enabled discovery of well-tolerated small-RNA carriers that could produce a therapeutic benefit in a challenging liver cancer model.Open in a separate windowFig. 1.RNA-based liver cancer therapies are hindered by the lack of potent and nontoxic delivery vehicles that avoid late-stage liver dysfunction that exacerbates toxicity. We envisioned that a modular design would allow discovery of dendrimers that balance low hepatotoxicity and high in vivo small-RNA delivery potency to tumor cells by fine-tuning of the identity and position of functional groups within dendrimer architectures.Efficacious delivery requires overcoming a series of extracellular and intracellular barriers (1). The physicochemical properties of small RNAs (high molecular weight, anionic charge, and hydrophilic nature) prevent passive diffusion across cell membranes. Studies of lipid and polymer carriers have implicated at least two common elements: chemical groups to bind small RNAs and nanoparticle (NP)-stabilizing hydrophobicity, which combine to encapsulate RNA molecules inside stable NPs and release small RNAs into the cytosol after endocytosis (35). Particular tertiary amines with optimal pK_a, alkyl chains, and defined topology/structure have been identified in effective carriers (2–10, 35–37).Despite these advances, demonstration of effective survival benefit in aggressive genetic models of human cancer has not been achieved with synthetic carriers due to the lack of delivery vehicles that avoid cancer-induced organ dysfunction. To address this, we designed a dendrimer library organized through binding, stabilization, and chemical functionality within the cores and peripheries. In vitro and in vivo evaluation identified dendrimers with optimal topological structures and high delivery efficacy (EC₅₀ of FVII knockdown < 0.02 mg/kg). Structure–activity knowledge was used to rationally design additional carriers with predicted in vivo activity. Evaluation of lead dendrimer candidates identified 5A2-SC8 as exhibiting low toxicity after repeated 75 mg/kg dendrimer i.v. dosing in chronically ill, tumor-bearing mice. Finally, we demonstrate that 5A2-SC8 can deliver a let-7g tumor suppressor miRNA with high in vivo potency to suppress tumor growth and result in a dramatic survival benefit of transgenic mice bearing aggressive, MYC-driven liver cancer. These findings suggest that modular degradable dendrimers may greatly expand the scope of chemical discovery research for therapeutic dendrimer delivery applications, ultimately providing new avenues for development of RNA cancer treatments by expansion of the therapeutic window.     

Autologous hematopoietic stem cell transplantation in adult acute lymphoblastic leukemia: still not out of fashion

The role of autologous hematopoietic stem cell transplantation (autoHSCT) in adult acute lymphoblastic leukemia (ALL) is still unclear. We retrospectively analyzed the results of the autoHSCT and maintenance therapy, with oral 6-mercaptopurine and methotrexate, in comparison to conventional-dose chemotherapy in the consolidation treatment of adult ALL and lymphoblastic lymphoma (LBL). The patients, with HLA identical sibling donor, underwent allogeneic transplantation, while the others were treated with autoHSCT and maintenance therapy with oral 6-mercaptopurine and methotrexate, or by conventional-dose chemotherapy (patient’s decision, no autologous hematopoietic stem cells harvest). Sixty consecutive adult patients (median age 35.2 years; range 17.3 to 70.7) with ALL (n = 52), LBL (n = 7), and acute biphenotypic leukemia (n = 1) were treated in our center from 1997 to 2007. Patients treated with chemotherapy alone (n = 35) had a shorter median progression-free survival (PFS) compared to patients who underwent autoHSCT plus maintenance therapy (n = 18), 8.4 and 46.8 months, respectively (p = 0.017). Patients treated with chemotherapy alone had also a shorter median overall survival (OS) compared to patients treated with autoHSCT: 13.0 vs. 46.8 months (p = 0.046). The differences remained statistically significant even after excluding patients with Ph positivity. We can conclude that, in our case, autoHSCT followed by maintenance chemotherapy is a good option for adult patients with ALL and, in standard-risk and high-risk patients, provides more favorable OS and PFS rates compared to patients treated by chemotherapy alone. However, we are aware of the fact that our analysis may have been distorted by the fact that the analysis is retrospective, that treatment with autoHSCT was based on patient’s decision, and that chemotherapy may have been administered to negatively selected patients.     

Prevention of central venous catheter related infections with chlorhexidine gluconate impregnated wound dressings: a randomized controlled trial

The objective of the study was to evaluate the effectiveness of chlorhexidine-impregnated sponges for reducing catheter-related infections of central venous catheters inserted for cancer chemotherapy. The method used was a randomized, prospective, open, controlled clinical study (three-step group sequential analysis protocol). The patients were from two high dependency units at a university hospital undergoing chemotherapy for haematological or oncological malignancies requiring central venous catheters (CVCs) expected to remain in place for at least 5 days. Six hundred and one patients with 9,731 catheterization days were studied between January 2004 and January 2006. Patients admitted for chemotherapy received chlorhexidine and silver sulfadiazine-impregnated triple-lumen CVCs under standardized conditions and were randomized to the groups receiving a chlorhexidine gluconate-impregnated wound dressing or a standard sterile dressing. Daily routine included clinical assessment of the insertion site (swelling, pain, redness), temperature, white blood count and C-reactive protein. Catheters remained in place until they were no longer needed or when a CVC-related infection was suspected. Infection was confirmed with blood cultures via the catheter lumina and peripheral blood cultures according to the time-to-positivity method. Six hundred and one patients were included. The groups were comparable with respect to demographic and clinical data. The incidence of CVC-related infections were 11.3% (34 of 301) and 6.3% (19 of 300) in the control and chlorhexidine-impregnated wound dressing groups, respectively (p = 0.016, relative risk 0.54; confidence interval 0.31–0.94). Especially, catheter-related infections at internal jugular vein insertions could be reduced (p = 0.018). No adverse effects related to the intervention were observed. The use of chlorhexidine-impregnated wound dressings significantly reduced the incidence of CVC-related infections in patients receiving chemotherapy.     

Presence of connexin 43 in subsarcolemmal, but not in interfibrillar cardiomyocyte mitochondria

Cardiomyocytes contain subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria, which differ in their respiratory and calcium retention capacity. Connexin 43 (Cx43) is located at the inner membrane of SSM, and Cx43 is involved in the cardioprotection by ischemic preconditioning (IP). The function of Cx43-formed channels is regulated in part by phosphorylation at residues in the carboxy terminus of Cx43. The aim of the present study was (1) to investigate whether Cx43 is also present in IFM, and (2) to characterize its spatial orientation in the inner mitochondrial membrane (IMM). Confirming previous findings, ADP-stimulated respiration was greater in IFM than in SSM from rat ventricles. In preparations from rats and mice not contaminated with sarcolemmal proteins, Cx43 was exclusively detected in SSM, but not in IFM by Western blot analysis (n = 6). SSM were exposed to different proteinase K concentrations to cleave peptide bonds, and Western blot analysis was performed for ATP synthase α (IMM, subunit in the matrix), uncoupling protein 3 (UCP3, IMM, intermembrane space epitope), and manganese superoxide dismutase (MnSOD, matrix). At a proteinase K concentration of 50 μg/ml, immunoreactivities of all the analyzed proteins were completely lost. The use of 5 μg/ml proteinase K resulted in similarly reduced immunoreactivities for Cx43 (19.4 ± 5.8% of untreated mitochondria, n = 6) and UCP3 (23.0 ± 4%, n = 7), whereas the immunoreactivities of ATP synthase α (49.1 ± 6.4%, n = 7) and MnSOD (79.9 ± 17.4%, n = 6) were better preserved, suggesting that the carboxy terminus of Cx43 is directed towards the intermembrane space. The results were confirmed in digitonin-treated mitochondria. Taken together, Cx43 is exclusively localized in SSM, with its carboxy terminus directed towards the intermembrane space. Since loss of mitochondrial Cx43 abolishes IP’s cardioprotection, SSM and IFM apparently differ in their function in the signal transduction of IP. K. Boengler and S. Stahlhofen contributed equally to this work.     

A Potential Shift From Adaptive Immune Activity to Nonspecific Inflammatory Activation Associated With Higher Depression Symptoms in Chronic Heart Failure Patients

BackgroundChronic heart failure (CHF) patients with elevated depression symptoms are at greater risk of morbidity and mortality. The mechanisms linking symptoms of depression with disease progression in CHF are unclear. However, research studies have found evidence of alterations in immune activity associated with depression symptoms that may influence heart function. The present study sought to determine the relationship between depression symptoms and chemotaxis of peripheral blood mononuclear cells (PBMCs) in CHF patients, both at rest and in response to moderate exercise.Methods and ResultsSixty-five patients diagnosed with CHF (mean age, 59.8 ± 14.5 years) and 45 non-CHF control subjects (mean age, 52.1 ± 11.6) completed the Beck Depression Inventory (BDI) before undergoing a moderate 20-minute bicycle exercise task. Chemotaxis of PBMCs was examined in vitro to a bacterial peptide f-met leu phe (fMLP) and a physiologic chemokine, stromal cell derived factor-1 (SDF-1) immediately before and after exercise. CHF patients had reduced chemotaxis to SDF-1 (P = .025) compared with non-CHF subjects. Higher BDI scores were associated with reduced baseline chemotaxis to SDF-1 in both CHF and non-CHF subjects (P = .027). In contrast, higher BDI scores were associated with increased chemotaxis to fMLP (P = .049) and SDF-1 (P = .018) in response to exercise in the CHF patients.ConclusionThe present study suggests a shift in immune cell mobility in CHF patients with greater depression symptom severity, with reduced chemotaxis to a physiologically specific chemokine at rest but increased chemotaxis to both nonspecific and specific chemical attractants in response to physical activity. This could have implications for cardiac repair and remodeling in CHF patients and therefore may affect disease progression.     

Pituitary-hormone secretion by thyrotropinomas

Hormone secretion by somatotropinomas, corticotropinomas and prolactinomas exhibits increased pulse frequency, basal and pulsatile secretion, accompanied by greater disorderliness. Increased concentrations of growth hormone (GH) or prolactin (PRL) are observed in about 30% of thyrotropinomas leading to acromegaly or disturbed sexual functions beyond thyrotropin (TSH)-induced hyperthyroidism. Regulation of non-TSH pituitary hormones in this context is not well understood. We there therefore evaluated TSH, GH and PRL secretion in 6 patients with up-to-date analytical and mathematical tools by 24-h blood sampling at 10-min intervals in a clinical research laboratory. The profiles were analyzed with a new deconvolution method, approximate entropy, cross-approximate entropy, cross-correlation and cosinor regression. TSH burst frequency and basal and pulsatile secretion were increased in patients compared with controls. TSH secretion patterns in patients were more irregular, but the diurnal rhythm was preserved at a higher mean with a 2.5 h phase delay. Although only one patient had clinical acromegaly, GH secretion and IGF-I levels were increased in two other patients and all three had a significant cross-correlation between the GH and TSH. PRL secretion was increased in one patient, but all patients had a significant cross-correlation with TSH and showed decreased PRL regularity. Cross-ApEn synchrony between TSH and GH did not differ between patients and controls, but TSH and PRL synchrony was reduced in patients. We conclude that TSH secretion by thyrotropinomas shares many characteristics of other pituitary hormone-secreting adenomas. In addition, abnormalities in GH and PRL secretion exist ranging from decreased (joint) regularity to overt hypersecretion, although not always clinically obvious, suggesting tumoral transformation of thyrotrope lineage cells.