Role of Rho-kinase in mediating contraction of chicken embryo femoral arteries

Rho-kinase-dependent Ca²⁺ sensitization is an essential process for contraction of mammalian vascular smooth muscle but the information about its effects in non-mammalian vessels is scarce. We aimed to investigate, using the Rho-kinase inhibitor hydroxyfasudil, the potential role of the Rho-kinase pathway of Ca²⁺ sensitization in depolarization- and agonist-mediated contraction of chicken embryo (at day 19 of the 21 days of incubation) femoral arteries. Contraction elicited by KCl (125 mM) comprised two phases (phasic and tonic contraction), both of which were abolished in the absence of extracellular Ca²⁺. Hydroxyfasudil (10 μM) left the initial phasic component nearly intact but abolished the tonic component. Hydroxyfasudil also induced a marked impairment of the contractions elicited by phenylephrine (PE), the thromboxane A₂ mimetic U46619, and endothelin-1. In contrast, inhibition of protein kinase C (PKC) by chelerythrine did not affect KCl- or PE-induced contractions, indicating lack of participation of PKC-mediated Ca²⁺ sensitization. Incubation under chronic hypoxia (15% O₂ from day 0) impaired embryonic growth but did not significantly affect hydroxyfasudil-mediated relaxation. In summary, our findings are indicative of a role for Rho-kinase activity in depolarization- and agonist-induced force generation in chicken embryo femoral arteries.     

Bi-functional cytokine fusion proteins for gene therapy and antibody-targeted treatment of cancer

Typically, multiple cytokines act in concert to mediate a desired immunological response, and thus more effective therapeutics may be achieved by combining several cytokines with potentially synergistic activities. We have developed a series of bi-functional cytokine fusion proteins which, when additionally linked to an intact antibody (or the Fc portion of an antibody) in a variety of configurations, can be specifically targeted. We focus here mainly on the synergizing cytokine combination interleukin-2/interleukin-12 (IL-2/IL-12), but also demonstrate the utility of this approach with interleukin-4/granulocyte-macrophage colony-stimulating factor (IL-4/GM-CSF). Cytokine activity was retained in constructs where the cytokines were fused in tandem at the carboxyl terminus of the Fc or antibody heavy (H) chain, as well as in constructs where one cytokine was fused at the carboxyl terminus of the H chain while the second cytokine was fused to the amino terminus of either the H or light (L) chain variable region. Even in such constructs, antigen binding of the antibody-cytokine fusion proteins could be maintained. In the context of bi-functional fusion proteins, hetero-dimeric IL-12 could be expressed either in a single-chain form, or maintained as a heterodimer in which the p40 subunit was fused to IL-2. These IL-12/IL-2 bi-functional fusion proteins were shown to induce extremely high levels of interferon-gamma (IFN-gamma), similar to the synergy normally seen with the combined application of the individual cytokines. In addition, these bifunctional molecules were shown to have striking anti-tumor activity as either gene therapy or as an antibody cytokine(s) fusion protein, and may provide a useful approach to the treatment of cancer.     

PML Nuclear Bodies and SATB1 Are Associated with HLA Class I Expression in EBV+ Hodgkin Lymphoma

Tumor cells of classical Hodgkin lymphoma (cHL) are characterized by a general loss of B cell phenotype, whereas antigen presenting properties are commonly retained. HLA class I is expressed in most EBV+ cHL cases, with an even enhanced expression in a proportion of the cases. Promyelocytic leukemia protein (PML) and special AT-rich region binding protein 1 (SATB1) are two global chromatin organizing proteins that have been shown to regulate HLA class I expression in Jurkat cells. We analyzed HLA class I, number of PML nuclear bodies (NBs) and SATB1 expression in tumor cells of 54 EBV+ cHL cases and used 27 EBV− cHL cases as controls. There was a significant difference in presence of HLA class I staining between EBV+ and EBV− cases (p<0.0001). We observed normal HLA class I expression in 35% of the EBV+ and in 19% of the EBV− cases. A stronger than normal HLA class I expression was observed in approximately 40% of EBV+ cHL and not in EBV− cHL cases. 36 EBV+ cHL cases contained less than 10 PML-NBs per tumor cell, whereas 16 cases contained more than 10 PML-NBs. The number of PML-NBs was positively correlated to the level of HLA class I expression (p<0.01). The percentage of SATB1 positive cells varied between 0% to 100% in tumor cells and was inversely correlated with the level of HLA class I expression, but only between normal and strong expression (p<0.05). Multivariable analysis indicated that the number of PML-NBs and the percentage of SATB1+ tumor cells are independent factors affecting HLA class I expression in EBV+ cHL. In conclusion, both PML and SATB1 are correlated to HLA class I expression levels in EBV+ cHL.     

Cyclic AMP induces integrin-mediated cell adhesion through Epac and Rap1 upon stimulation of the beta 2-adrenergic receptor

cAMP controls many cellular processes mainly through the activation of protein kinase A (PKA). However, more recently PKA-independent pathways have been established through the exchange protein directly activated by cAMP (Epac), a guanine nucleotide exchange factor for the small GTPases Rap1 and Rap2. In this report, we show that cAMP can induce integrin-mediated cell adhesion through Epac and Rap1. Indeed, when Ovcar3 cells were treated with cAMP, cells adhered more rapidly to fibronectin. This cAMP effect was insensitive to the PKA inhibitor H-89. A similar increase was observed when the cells were transfected with Epac. Both the cAMP effect and the Epac effect on cell adhesion were abolished by the expression of Rap1-GTPase-activating protein, indicating the involvement of Rap1 in the signaling pathway. Importantly, a recently characterized cAMP analogue, 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate, which specifically activates Epac but not PKA, induced Rap-dependent cell adhesion. Finally, we demonstrate that external stimuli of cAMP signaling, i.e., isoproterenol, which activates the G alpha s-coupled beta 2-adrenergic receptor can induce integrin-mediated cell adhesion through the Epac-Rap1 pathway. From these results we conclude that cAMP mediates receptor-induced integrin-mediated cell adhesion to fibronectin through the Epac-Rap1 signaling pathway.     

Cutting edge: resistance to HIV-1 infection among African female sex workers is associated with inhibitory KIR in the absence of their HLA ligands

NK cells are regulated in part by killer Ig-like receptors (KIR) that interact with HLA molecules on potential target cells. KIR and HLA loci are highly polymorphic and certain KIR/HLA combinations were found to protect against HIV disease progression. We show in this study that KIR/HLA interactions also influence resistance to HIV transmission. HIV-exposed but seronegative female sex workers in Abidjan, C?te d'Ivoire, frequently possessed inhibitory KIR genes in the absence of their cognate HLA genes: KIR2DL2/KIR2DL3 heterozygosity in the absence of HLA-C1 and KIR3DL1 homozygosity in the absence of HLA-Bw4. HIV-seropositive female sex workers were characterized by corresponding inhibitory KIR/HLA pairings: KIR2DL3 homozygosity together with HLA-C1 and a trend toward KIR3DL1/HLA-Bw4 homozygosity. Absence of ligands for inhibitory KIR could lower the threshold for NK cell activation. In addition, exposed seronegatives more frequently possessed AB KIR genotypes, which contain more activating KIR. The data support an important role for NK cells and KIR/HLA interactions in antiviral immunity.     

Bats and birds increase crop yield in tropical agroforestry landscapes

Human welfare is significantly linked to ecosystem services such as the suppression of pest insects by birds and bats. However, effects of biocontrol services on tropical cash crop yield are still largely unknown. For the first time, we manipulated the access of birds and bats in an exclosure experiment (day, night and full exclosures compared to open controls in Indonesian cacao agroforestry) and quantified the arthropod communities, the fruit development and the final yield over a long time period (15 months). We found that bat and bird exclusion increased insect herbivore abundance, despite the concurrent release of mesopredators such as ants and spiders, and negatively affected fruit development, with final crop yield decreasing by 31% across local (shade cover) and landscape (distance to primary forest) gradients. Our results highlight the tremendous economic impact of common insectivorous birds and bats, which need to become an essential part of sustainable landscape management.     

Anthropometric factors, physical activity, and risk of non-Hodgkin's lymphoma in the Women's Health Initiative

Background: Incidence rates of non-Hodgkin's lymphoma (NHL) increased substantially in the United States and worldwide during the latter part of the 20th century, but little is known about its etiology. Obesity is associated with impaired immune function through which it may influence the risk of NHL; other factors reflecting energy homeostasis (height, abdominal adiposity, and physical activity) may also be involved. Methods: We examined the association of anthropometric factors and physical activity with risk of NHL and its major subtypes in a large cohort of women aged 50–79 years old who were enrolled at 40 clinical centers in the United States between 1993 and 1998. Over a mean follow-up period of 11 years, 1123 cases of NHL were identified among 158,975 women. Cox proportional hazards models were used to estimate hazard ratios (HR) and 95% confidence intervals (CI). Results: Height at baseline was positively associated with risk of all NHL and with that of diffuse large B-cell lymphoma (HRs_q4vs.q1 1.19, 95% CI 1.00–1.43 and 1.43, 95% CI 1.01–2.03, respectively). Measures of obesity and abdominal adiposity at baseline were not associated with risk. Hazard ratios for NHL were increased for women in the highest quartile of weight and body mass index at age 18 (HRs_q4vs.q1 1.29, 95% CI 1.01–1.65 and 1.27, 95% CI 1.01–1.59, respectively). Some measures of recreational physical activity were modestly associated with increased risk of NHL overall, but there were no clear associations with specific subtypes. Conclusion: Our findings regarding anthropometric measures are consistent with those of several previous reports, suggesting that early life influences on growth and immune function may influence the risk of NHL later in life.