Inhibitory effects of ethacrynic acid analogues lacking the α,β-unsaturated carbonyl unit and para-acylated phenols on human cancer cells

A series of ethacrynic acid analogues, lacking the α,β-unsaturated carbonyl unit, was synthesized and subsequently evaluated for their ability to inhibit the migration of human breast cancer cells, Hs578Ts(i)8 as well as of human prostate cancer cells, C4-2B. These cell lines provide a good model system to study migration and invasion, since they represent metastatic cancer. Our studies show that ethacrynic acid analogues with methyl substituents at the aromatic ring demonstrate no inhibitory effect on the migration of both cancer cell lines, whereas a precursor in the synthesis of these ethacrynic acid analogues (II-1, a para-acylated m-cresol) is an excellent inhibitor of the migration of both cancer cell lines.     

Interleukin-15 but not interleukin-7 abrogates vaccine-induced decrease in virus level in simian immunodeficiency virus mac251-infected macaques

The loss of CD4(+) T cells and the impairment of CD8(+) T cell function in HIV infection suggest that pharmacological treatment with IL-7 and IL-15, cytokines that increase the homeostatic proliferation of T cells and improve effector function, may be beneficial. However, these cytokines could also have a detrimental effect in HIV-1-infected individuals, because both cytokines increase HIV replication in vitro. We assessed the impact of IL-7 and IL-15 treatment on viral replication and the immunogenicity of live poxvirus vaccines in SIV(mac251)-infected macaques (Macaca mulatta). Neither cytokine augmented the frequency of vaccine-expanded CD4(+) or CD8(+) memory T cells, clonal recruitment to the SIV-specific CD8(+) T cell pool, or CD8(+) T cell function. Vaccination alone transiently decreased the viral set point following antiretroviral therapy suspension. IL-15 induced massive proliferation of CD4(+) effector T cells and abrogated the ability of vaccination to decrease set point viremia. In contrast, IL-7 neither augmented nor decreased the vaccine effect and was associated with a decrease in TGF-beta expression. These results underscore the importance of testing immunomodulatory approaches in vivo to assess potential risks and benefits for HIV-1-infected individuals.     

Differential distribution of proteins and lipids in detergent-resistant and detergent-soluble domains in rod outer segment plasma membranes and disks

Membrane heterogeneity plays a significant role in regulating signal transduction and other cellular activities. We examined the protein and lipid components associated with the detergent-resistant membrane (DRM) fractions from retinal rod outer segment (ROS) disk and plasma membrane-enriched preparations. Proteomics and correlative western blot analysis revealed the presence of α and β subunits of the rod cGMP-gated ion channel and glucose transporter type 1, among other proteins. The glucose transporter was present exclusively in ROS plasma membrane (not disks) and was highly enriched in DRMs, as was the cGMP-gated channel β-subunit. In contrast, the majority of rod opsin and ATP-binding cassette transporter A4 was localized to detergent-soluble domains in disks. As expected, the cholesterol : fatty acid mole ratio was higher in DRMs than in the corresponding parent membranes (disk and plasma membranes, respectively) and was also higher in disks compared to plasma membranes. Furthermore, the ratio of saturated : polyunsaturated fatty acids was also higher in DRMs compared to their respective parent membranes (disk and plasma membranes). These results confirm that DRMs prepared from both disks and plasma membranes are enriched in cholesterol and in saturated fatty acids compared to their parent membranes. The dominant fatty acids in DRMs were 16 : 0 and 18 : 0; 22 : 6n3 and 18 : 1 levels were threefold higher and twofold lower, respectively, in disk-derived DRMs compared to plasma membrane-derived DRMs. We estimate, based on fatty acid recovery that DRMs account for only ∼ 8% of disks and ∼ 12% of ROS plasma membrane.     

Prolonged blockade of VEGF family members does not cause identifiable damage to retinal neurons or vessels

Several ocular diseases complicated by neovascularization are being treated by repeated intraocular injections of vascular endothelial growth factor (VEGF) antagonists. While substantial benefits have been documented, there is concern that unrecognized damage may be occurring, because blockade of VEGF may damage the fenestrated vessels of the choroicapillaris and deprive retinal neurons of input from a survival factor. One report has suggested that even temporary blockade of all isoforms of VEGF-A results in significant loss of retinal ganglion cells. In this study, we utilized double transgenic mice with doxycycline-inducible expression of soluble VEGF receptor 1 coupled to an Fc fragment (sVEGFR1Fc), a potent antagonist of several VEGF family members, including VEGF-A, to test the effects of VEGF blockade in the retina. Expression of sVEGFR1Fc completely blocked VEGF-induced retinal vascular permeability and significantly suppressed the development of choroidal neovascularization at rupture sites in Bruch's membrane, but did not cause regression of established choroidal neovascularization. Mice with constant expression of sVEGFR1Fc in the retina for 7 months had normal electroretinograms and normal retinal and choroidal ultrastructure including normal fenestrations in the choroicapillaris. They also showed no significant difference from control mice in the number of ganglion cell axons in optic nerve cross sections and the retinal level of mRNA for 3 ganglion cell-specific genes. These data indicate that constant blockade of VEGF for up to 7 months has no identifiable deleterious effects on the retina or choroid and support the use of VEGF antagonists in the treatment of retinal diseases.     

Cutting edge: expression of TNFR2 defines a maximally suppressive subset of mouse CD4+CD25+FoxP3+ T regulatory cells: applicability to tumor-infiltrating T regulatory cells

TNFR2 is predominantly expressed by a subset of human and mouse CD4(+)CD25(+)FoxP3(+) T regulatory cells (Tregs). In this study, we characterized the phenotype and function of TNFR2(+) Tregs in peripheral lymphoid tissues of normal and tumor-bearing C57BL/6 mice. We found that TNFR2 was expressed on 30-40% of the Tregs of the peripheral activated/memory subset that were most highly suppressive. In contrast, TNFR2(-) Tregs exhibited the phenotype of naive cells and only had minimal suppressive activity. Although not typically considered to be Tregs, CD4(+)CD25(-)TNFR2(+) cells nevertheless possessed moderate suppressive activity. Strikingly, the suppressive activity of TNFR2(+) Tregs was considerably more potent than that of reportedly highly suppressive CD103(+) Tregs. In the Lewis lung carcinoma model, more highly suppressive TNFR2(+) Tregs accumulated intratumorally than in the periphery. Thus, TNFR2 identifies a unique subset of mouse Tregs with an activated/memory phenotype and maximal suppressive activity that may account for tumor-infiltrating lymphocyte-mediated immune evasion by tumors.     

Chemotactic activity of S100A7 (Psoriasin) is mediated by the receptor for advanced glycation end products and potentiates inflammation with highly homologous but functionally distinct S100A15

Human S100A7 (psoriasin) is overexpressed in inflammatory diseases. The recently discovered, co-evolved hS100A15 is almost identical in sequence and up-regulated with hS100A7 during cutaneous inflammation. The functional role of these closely related proteins for inflammation remains undefined. By generating specific Abs, we demonstrate that hS100A7 and hS100A15 proteins are differentially expressed by specific cell types in the skin. Although highly homologous, both proteins are chemoattractants with distinct chemotactic activity for leukocyte subsets. We define RAGE (receptor for advanced glycation end products) as the hS100A7 receptor, whereas hS100A15 functions through a Gi protein-coupled receptor. hS100A7-RAGE binding, signaling, and chemotaxis are zinc-dependent in vitro, reflecting the previously reported zinc-mediated changes in the hS100A7 dimer structure. When combined, hS100A7 and hS100A15 potentiate inflammation in vivo. Thus, proinflammatory synergism in disease may be driven by the diverse biology of these almost identical proteins that have just recently evolved. The identified S100A7 interaction with RAGE may provide a novel therapeutic target for inflammation.     

RANKL inhibition by osteoprotegerin prevents bone loss without affecting local or systemic inflammation parameters in two rat arthritis models: comparison with anti-TNFα or anti-IL-1 therapies

Introduction

Rat adjuvant-induced arthritis (AIA) and collagen-induced arthritis (CIA) feature bone loss and systemic increases in TNFα, IL-1β, and receptor activator of NF-κB ligand (RANKL). Anti-IL-1 or anti-TNFα therapies consistently reduce inflammation in these models, but systemic bone loss often persists. RANKL inhibition consistently prevents bone loss in both models without reducing joint inflammation. Effects of these therapies on systemic markers of bone turnover and inflammation have not been directly compared.

Methods

Lewis rats with established AIA or CIA were treated for 10 days (from day 4 post onset) with either PBS (Veh), TNFα inhibitor (pegsunercept), IL-1 inhibitor (anakinra), or RANKL inhibitor (osteoprotegerin (OPG)-Fc). Local inflammation was evaluated by monitoring hind paw swelling. Bone mineral density (BMD) of paws and lumbar vertebrae was assessed by dual X-ray absorptiometry. Markers and mediators of bone resorption (RANKL, tartrate-resistant acid phosphatase 5b (TRACP 5B)) and inflammation (prostaglandin E₂ (PGE₂), acute-phase protein alpha-1-acid glycoprotein (α₁AGP), multiple cytokines) were measured in serum (day 14 post onset).

Results

Arthritis progression significantly increased paw swelling and ankle and vertebral BMD loss. Anti-TNFα reduced paw swelling in both models, and reduced ankle BMD loss in AIA rats. Anti-IL-1 decreased paw swelling in CIA rats, and reduced ankle BMD loss in both models. Anti-TNFα and anti-IL-1 failed to prevent vertebral BMD loss in either model. OPG-Fc reduced BMD loss in ankles and vertebrae in both models, but had no effect on paw swelling. Serum RANKL was elevated in AIA-Veh and CIA-Veh rats. While antiTNFα and anti-IL-1 partially normalized serum RANKL without any changes in serum TRACP 5B, OPG-Fc treatment reduced serum TRACP 5B by over 90% in both CIA and AIA rats. CIA-Veh and AIA-Veh rats had increased serum α₁AGP, IL-1β, IL-8 and chemokine (C-C motif) ligand 2 (CCL2), and AIA-Veh rats also had significantly greater serum PGE₂, TNFα and IL-17. Anti-TNFα reduced systemic α₁AGP, CCL2 and PGE₂ in AIA rats, while anti-IL-1 decreased systemic α₁AGP, IL-8 and PGE₂. In contrast, RANKL inhibition by OPG-Fc did not lessen systemic cytokine levels in either model.

Conclusions

Anti-TNFα or anti-IL-1 therapy inhibited parameters of local and systemic inflammation, and partially reduced local but not systemic bone loss in AIA and CIA rats. RANKL inhibition prevented local and systemic bone loss without significantly inhibiting local or systemic inflammatory parameters.     

INTRODUCTION

The Oak Spring Garden Library, a legacy of Rachel Lambert Mellon (1910–2014) and now part of the Oak Spring Garden Foundation, contains the archive of John Bradby Blake (1745–1773), a young trader who worked for the British East India Company in the late 1760s and early 1770s in Canton (Guangzhou) and Macau. The archive provides an unusually detailed insight into Bradby Blake's life and botanical work, including his attempts to send living plants back to England. The archive also contains almost 200 pieces of exquisitely detailed early British‐Chinese hybrid botanical art, which Bradby Blake prepared with a Chinese artist, Mauk‐Sow‐U. With their emphasis on botanical detail and careful depiction of the detailed structure of flowers, these illustrations are as accurate as any produced at this relatively early date in the post‐Linnaean development of botanical science.