Search

EffeCtiveness of biologic treAtmeNts for plaque psOriasis in Italy: An obserVAtional (CANOVA) study was aimed at providing real-world evidence of the effectiveness of biologics in Italian patients with moderate–severe psoriasis. It was an observational, retro-prospective cohort study conducted in 17 Italian dermatology clinics. Adult patients with moderate–severe plaque psoriasis, who started a biologic treatment between 24 weeks and 24 months before enrolment, were included. With a follow-up visit at 6months after enrolment, each patient had at least 12 months of observation. The primary objective was to describe the clinical response rates (PASI 75) after 16/24/52 weeks from biologic treatment start. Secondary outcomes were sustained response, quality of life, and treatment satisfaction. Of the 669 eligible patients (64% males), 52% were naïve to biologics, though a mean duration of psoriasis since first diagnosis of 18.6 years (SD 13.2). The most frequently prescribed biologics were secukinumab (41%), ustekinumab (25%), TNF-inhibitors (22%) and ixekizumab (12%). PASI 75 was achieved by 86% of patients (95% CI: 82%–89%) at 16 weeks, 90% (87%–93%) at 24 weeks, and 91% (89%–94%) at 52 weeks. Patients achieving PASI 90 and PASI 100 at 52 weeks were 75% (71%–79%) and 53% (49%–57%), respectively. Sustained PASI 75 response after 1year from treatment start was achieved by 78% (74%–82%) of patients. Mean DLQI total score was 2.3 (SD 3.9) at enrollment and decreased at the final visit to 1.8 (3.6). A high level of treatment satisfaction was expressed by patients over the study period. This large real-world study confirms in the clinical practice the good effectiveness and acceptability of biologics in psoriasis patients.

CD13/aminopeptidase N is a multifunctional cell surface metalloprotease expressed in myeloid and activated endothelial cells; in epithelial cells of the intestine, liver, and kidney; and in other cells. A growing body of evidence suggests that CD13 has an important role in inflammation and angiogenesis. Furthermore, a CD13 form selectively expressed by the angiogenic endothelium, but not in normal vessels, is recognized by peptides containing the Asn-Gly-Arg (NGR) motif. Because of this property, NGR peptides have been exploited as ligands for the selective delivery of a variety of therapeutic and imaging agents to angiogenic vessels. In this chapter, we discuss the structural and functional properties of NGR peptides and their potential applications as drug delivery systems in diseases with angiogenesis and inflammatory components. In addition, we discuss the experimental evidence suggesting that the asparagine residue of NGR can rapidly undergo deamidation, a spontaneous reaction that changes NGR to isoDGR (an integrin-binding motif), and the potential biological, pharmacological, and toxicological implications of this sequence transition in peptide-drug conjugates.

// Flavio Curnis 1,* , Alice Dallatomasina 1,* , Mimma Bianco 1,* , Anna Gasparri 1 , Angelina Sacchi 1 , Barbara Colombo 1 , Martina Fiocchi 1 , Laura Perani 2 , Massimo Venturini 2 , Carlo Tacchetti 2 , Suvajit Sen 3 , Ricardo Borges 4 , Eleonora Dondossola 1 , Antonio Esposito 2,5 , Sushil K. Mahata 6 and Angelo Corti 1,5 1 Division of Experimental Oncology, IRCCS San Raffaele Scientific Institute, Milan, Italy 2 Experimental Imaging Center, IRCCS San Raffaele Scientific Institute, Milan, Italy 3 University of California, Los Angeles, CA, USA 4 La Laguna University, Tenerife, Spain 5 Vita-Salute San Raffaele University, Milan, Italy 6 VA San Diego Healthcare System and University of California, San Diego, La Jolla, CA, USA * These authors have contributed equally to this work Correspondence to: Flavio Curnis, email: // Angelo Corti, email: // Keywords : chromogranin A, angiogenesis, tumor perfusion, endothelial cells, protease-nexin-1 Received : September 9, 2016 Accepted : September 17, 2016 Published : September 24, 2016 Abstract Chromogranin A (CgA), a neuroendocrine secretory protein, and its fragments are present in variable amounts in the blood of normal subjects and cancer patients. We investigated whether circulating CgA has a regulatory function in tumor biology and progression. Systemic administration of full-length CgA, but not of fragments lacking the C-terminal region, could reduce tumor growth in murine models of fibrosarcoma, mammary adenocarcinoma, Lewis lung carcinoma, and primary and metastatic melanoma, with U-shaped dose-response curves. Tumor growth inhibition was associated with reduction of microvessel density and blood flow in neoplastic tissues. Neutralization of endogenous CgA with antibodies against its C-terminal region (residues 410-439) promoted tumor growth. Structure-function studies showed that the C-terminal region of CgA contains a bioactive site and that cleavage of this region causes a marked loss of anti-angiogenic and anti-tumor potency. Mechanistic studies showed that full-length CgA could induce, with a U-shaped dose-response curve, the production of protease nexin-1 in endothelial cells, a serine protease inhibitor endowed of anti-angiogenic activity. Gene silencing or neutralization of protease nexin-1 with specific antibodies abolished both anti-angiogenic and anti-tumor effects of CgA. These results suggest that circulating full-length CgA is an important inhibitor of angiogenesis and tumor growth, and that cleavage of its C-terminal region markedly reduces its activity. Pathophysiological changes in CgA blood levels and/or its fragmentation might regulate disease progression in cancer patients.

Colloidal gold (Au), a well-tolerated nanomaterial, is currently exploited for several applications in nanomedicine. We show that gold nanoparticles tagged with a novel tumor-homing peptide containing Asn-Gly-Arg (NGR), a ligand of CD13 expressed by the tumor neovasculature, can be exploited as carriers for cytokine delivery to tumors. Biochemical and functional studies showed that the NGR molecular scaffold/linker used for gold functionalization is critical for CD13 recognition. Using fibrosarcoma-bearing mice, NGR-tagged nanodrugs could deliver extremely low, yet pharmacologically active doses of tumor necrosis factor (TNF), an anticancer cytokine, to tumors with no evidence of toxicity. Mechanistic studies confirmed that CD13 targeting was a primary mechanism of drug delivery and excluded a major role of integrin targeting consequent to NGR deamidation, a degradation reaction that generates the isoAsp-Gly-Arg (isoDGR) integrin ligand. NGR-tagged gold nanoparticles can be used, in principle, as a novel platform for single- or multi-cytokine delivery to tumor endothelial cells for cancer therapy.

NGR (asparagine-glycine-arginine) is a tumor vasculature-homing peptide motif widely used for the functionalization of drugs, nanomaterials and imaging compounds for cancer treatment and diagnosis. Unfortunately, this motif has a strong propensity to undergo rapid deamidation. This reaction, which converts NGR into isoDGR, is associated with receptor switching from CD13 to integrins, with potentially important manufacturing, pharmacological and toxicological implications. It is found that glycine N-methylation of NGR-tagged nanocarriers completely prevents asparagine deamidation without impairing CD13 recognition. Studies in animal models have shown that the methylated NGR motif can be exploited for delivering radiolabeled compounds and nanocarriers, such as tumor necrosis factor-α (TNF)-bearing nanogold and liposomal doxorubicin, to tumors with improved selectivity. These findings suggest that this NGR derivative is a stable and efficient tumor-homing ligand that can be used for delivering functional nanomaterials to tumor vasculature.

HTLV-1 orf-I is linked to immune evasion, viral replication and persistence. Examining the orf-I sequence of 160 HTLV-1-infected individuals; we found polymorphism of orf-I that alters the relative amounts of p12 and its cleavage product p8. Three groups were identified on the basis of p12 and p8 expression: predominantly p12, predominantly p8 and balanced expression of p12 and p8. We found a significant association between balanced expression of p12 and p8 with high viral DNA loads, a correlate of disease development. To determine the individual roles of p12 and p8 in viral persistence, we constructed infectious molecular clones expressing p12 and p8 (D26), predominantly p12 (G29S) or predominantly p8 (N26). As we previously showed, cells expressing N26 had a higher level of virus transmission in vitro. However, when inoculated into Rhesus macaques, cells producing N26 virus caused only a partial seroconversion in 3 of 4 animals and only 1 of those animals was HTLV-1 DNA positive by PCR. None of the animals exposed to G29S virus seroconverted or had detectable viral DNA. In contrast, 3 of 4 animals exposed to D26 virus seroconverted and were HTLV-1 positive by PCR. In vitro studies in THP-1 cells suggested that expression of p8 was sufficient for productive infection of monocytes. Since orf-I plays a role in T-cell activation and recognition; we compared the CTL response elicited by CD4+ T-cells infected with the different HTLV-1 clones. Although supernatant p19 levels and viral DNA loads for all four infected lines were similar, a significant difference in Tax-specific HLA.A2-restricted killing was observed. Cells infected with Orf-I-knockout virus (12KO), G29S or N26 were killed by CTLs, whereas cells infected with D26 virus were resistant to CTL killing. These results indicate that efficient viral persistence and spread require the combined functions of p12 and p8., Author Summary HTLV-1 persists despite a vigorous host immune response. We found that polymorphism of HTLV-1 orf-I alter the relative amounts of the p12 precursor and its cleavage product p8, and is associated with differences in blood virus levels in humans, a correlate of disease risk. Reverse genetics in 160 HTLV-1 infected individuals demonstrated that equivalent levels of p8 and p12 are associated with high virus levels and, accordingly, genetically engineered HTLV-1s that express either predominantly p12 or p8 are poorly infectious in macaques. We found that expression of p8 is sufficient for productive infection of monocytes. Expression of either p12 alone or p8 alone is insufficient to protect infected cells from MHC-class-I restricted CTL killing. However, the balanced expression of both provides resistance of infected cells to CTL killing. Together, our findings provide the rationale to explore novel approaches to target the cleavage of the p12 protein, an essential step for viral infectivity and persistence.

The human T-cell leukemia/lymphoma virus type 1 (HTLV-1) p30 protein, essential for virus infectivity in vivo , is required for efficient infection of human dendritic cells (DCs) but not B and T cells in vitro . We used a human monocytic cell line, THP-1, and dendritic cells to study the mechanism of p30 and p12/p8 requirements in these cell types. p30 inhibited the expression of interferon (IFN)-responsive genes (ISG) following stimulation by lipopolysaccharide (LPS) of Toll-like receptor 4 (TLR4) and by poly(I·C) of TLR3 but not of TLR7/8 with imiquimod. Results with THP-1 mirrored those for ex vivo human primary monocytes and monocyte-derived dendritic cells (Mo-mDC). The effect of p30 on TLR signaling was also demonstrated by ablating its expression within a molecular clone of HTLV-1. HTLV-1 infection of monocytes inhibited TLR3- and TLR4-induced ISG expression by 50 to 90% depending on the genes, whereas the isogenic clone p30 knockout virus was less effective at inhibiting TLR3 and TRL4 signaling and displayed lower infectivity. Viral expression and inhibition of ISG transcription was, however, rescued by restoration of p30 expression. A chromatin immunoprecipitation assay demonstrated that p30 inhibits initiation and elongation of PU.1-dependent transcription of IFN-α1, IFN-β, and TLR4 genes upon TLR stimulation. In contrast, experiments conducted with p12/p8 did not demonstrate an effect on ISG expression. These results provide a mechanistic explanation of the requirement of p30 for HTLV-1 infectivity in vivo , suggest that dampening interferon responses in monocytes and DCs is specific for p30, and represent an essential early step for permissive HTLV-1 infection and persistence.

Colloidal gold (Au) is a well-tolerated nanomaterial currently exploited for several applications in the field of nanomedicine. In particular, gold nanoparticles loaded with tumor necrosis factor-alpha (TNF), a cytokine endowed with anti tumor activity, have a therapeutic index higher than that of TNF. For this reason TNF-gold nanoparticles are currently tested in cancer patients. We have addressed the hypothesis that TNF-gold nanoparticles tagged with tumor vasculature homing peptides have improved therapeutic properties. To this aim we have tagged TNF-gold nanoparticles with albumin-peptide conjugates containing the NGR (Asn-Gly-Arg) or isoDGR (isoAsp-Gly-Arg) motives, i.e. with ligands of CD13 and integrin alphavbeta3, respectively, expressed by the tumor neovasculature. The therapeutic properties of the resulting nanodrugs (called NGR-HSA/Au/TNF and isoDGR-HSA/Au/TNF) were then investigated in fibrosarcoma- or lymphoma-bearing mice and compared with those a nanodrug lacking the targeting peptide (called HSA/Au/TNF). The results showed that intravenous administration of extremely low doses of NGR-HSA/Au/TNF or isoDGR-HSA/Au/TNF (equivalent to 5 pg of bioactive TNF/mouse) were sufficient to induce anti-tumor effects, whereas similar doses of HSA/Au/TNF were minimally active. Low-dose NGR-HSA/Au/TNF and isoDGR-HSA/Au/TNF, but not HSA/Au/TNF, could also exert synergistic anti-tumor effects with melphalan, a chemotherapeutic drug. These findings support the concept that tagging TNF-gold nanoparticles with tumor vasculature homing peptides have an improved antitumor activity, likely because of an active targeting mechanism. Citation Format: Flavio Curnis, AnnaMaria Gasparri, Angelina Sacchi, Martina Fiocchi, Angelo Corti. Anti-tumor activity of TNF-gold nanodrugs tagged with tumor vasculature-homing peptides containing the NGR or isoDGR motives. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4387. doi:10.1158/1538-7445.AM2015-4387

We have shown that HTLV-1 p30 is critical for cell-free infection of primary dendritic cells (DCs) and that knockout of p30 in the HTLV-1 molecular clone p30KO-HTLV-1 decreases infectivity in rhesus macaques. In addition, p30 was recently shown to influence TLR signaling and expression of cellular genes involved in apoptosis, cell-cycle and transcription, suggesting that p30 plays a role in innate immune responses. Using the monocytic cell line THP-1 transduced with a p30-expressing lentiviral vector, we found that p30 inhibits type-I interferon (IFN) responses. While p30 did not affect the basal expression of type-1 IFN stimulated genes (ISGs), a significant decrease (50-80%) in mRNA levels of MxA, OAS and A3G was found following stimulation of p30-transduced cells with either polyI:C or LPS. HTLV-1-p30 did not affect ISG induction following Imiquimod stimulation, indicating that p30 affects specific signaling pathways. Importantly, these results were confirmed in primary monocyte-derived DCs. Infection of THP-1 cells with wild type (WT) HTLV-1 inhibits ISGs expression 10-50% lower than infection with p30 knockout virus. Moreover, THP-1 cells infected with p30KO-HTLV-1 produced lower levels of virus than WT-HTLV-1. Co-expression of p30 in p30KO-HTLV-1-infected cells rescued the inhibition of ISGs transcription and viral production. ChIP assays revealed that expression of p30 inhibited transcription of IFNα1, IFNβ and TLR4 following TLR stimulation by inhibiting PU.1 promoter binding. These results demonstrate that p30 protein is important for establishing HTLV-1 infection by dampening the antiviral response elicited by monocytes/macrophages and DCs, allowing persistence, replication and spread of HTLV-1 infection.

In HTLV-1 endemic areas, 5 to 10% of HIV-1-infected individuals are also coinfected with HTLV-1/2. Several studies support the finding that dual infection of HIV-1 and HTLV-2 may confer delayed AIDS progression. In contrast, HTLV-1 has been concluded to have either no effect or to increase progression to AIDS in HIV-1-infected individuals. Although Tax-1 is known to influence HIV-1 replication in vitro, other HTLV-1 proteins likely have a role in retroviral coinfection. Here, we are investigating whether HTLV-1 proteins regulate activation of HIV-1 expression in latently-infected cells. In this study, we used B-cells infected with molecular clones that either express both p12 and p8, p12-only, p8-only, or contain knockouts to orf-I, orf-II, or hbz. The HTLV-1-infected B-cells were cocultured with KK1 T-cells latently-infected with HIV-1. Following coculture, cells and media were assayed for viral expression. Analyses of immunoblot and ELISA data suggest that HTLV-1-infected B-cells that express p8 enhanced activation of latent HIV-1 expression. We are currently examining whether cell-to-cell contact with p8 expressing cells is required to reactivate the latently-infected KK1 cells. Results from this study could have an impact on treatment of patients coinfected with HIV-1 and HTLV-1.