Cristiano Sacchetti

In vivo targeting of intratumor regulatory T cells using PEG-modified single-walled carbon nanotubes.

Recent evidence regarding the role of regulatory T cells (Treg) in tumor development has suggested that the manipulation of Treg function selectively in the tumor microenvironment would be a desirable immunotherapy approach. Targeting intratumor immune populations would reduce side effects on peripheral healthy cells and increase antitumor efficacy of immunotherapies. However, no current approaches are available which enable selective in vivo targeting of intratumor Treg or other immune cell subpopulations. Herein, we investigated the ability of ligands against Treg-specific receptors to drive selective internalization of PEG-modified single-walled carbon nanotubes (PEG-SWCNTs) into Treg residing in the tumor microenvironment. We focused our attention on the glucocorticoid-induced TNFR-related receptor (GITR), as it showed higher overexpression on intratumor vs peripheral (i.e., splenic) Treg compared to other reported Treg-specific markers (folate receptor 4, CD103, and CD39). Ex vivo investigations showed that the Treg targeting efficiency and selectivity of PEG-SWCNTs depended on incubation time, dose, number of ligands per nanotube, and targeted surface marker. In vivo investigations showed that PEG-SWCNTs armed with GITR ligands targeted Treg residing in a B16 melanoma more efficiently then intratumor non-Treg or splenic Treg. The latter result was achieved by exploiting a combination of passive tumor targeting due to enhanced tumor vascular permeability, naturally increased intratumor Treg vs effector T cell (Teff) ratio, and active targeting of markers that are enriched in intratumor vs splenic Treg. We also found that PEG-SWCNTs loaded with GITR ligands were internalized by Treg through receptor-mediated endocytosis and transported into the cytoplasm and nucleus ex vivo and in vivo. This is the first example of intratumor immune cell targeting and we hope it will pave the way to innovative immunotherapies against cancer.

Polyethylene-Glycol-Modified Single-Walled Carbon Nanotubes for Intra-Articular Delivery to Chondrocytes

Osteoarthritis (OA) is a common and debilitating degenerative disease of articular joints for which no disease-modifying medical therapy is currently available. Inefficient delivery of pharmacologic agents into cartilage-resident chondrocytes after systemic administration has been a limitation to the development of anti-OA medications. Direct intra-articular injection enables delivery of high concentrations of agents in close proximity to chondrocytes; however, the efficacy of this approach is limited by the fast clearance of small molecules and biomacromolecules after injection into the synovial cavity. Coupling of pharmacologic agents with drug delivery systems able to enhance their residence time and cartilage penetration can enhance the effectiveness of intra-articularly injected anti-OA medications. Herein we describe an efficient intra-articular delivery nanosystem based on single-walled carbon nanotubes (SWCNTs) modified with polyethylene glycol (PEG) chains (PEG-SWCNTs). We show that PEG-SWCNTs are capable to persist in the joint cavity for a prolonged time, enter the cartilage matrix, and deliver gene inhibitors into chondrocytes of both healthy and OA mice. PEG-SWCNT nanoparticles did not elicit systemic or local side effects. Our data suggest that PEG-SWCNTs represent a biocompatible and effective nanocarrier for intra-articular delivery of agents to chondrocytes.

Novel IgE Recognized Components of Lolium perenne Pollen Extract: Comparative Proteomics Evaluation of Allergic Patients Sensitization Profiles

In the last years, proteomic investigation provided a powerful tool in molecular characterization of complex allergen sources with relevant implications in both diagnosis and immunotherapic treatment of allergies. We followed a proteomic approach to characterize ryegrass (Lolium perenne) pollen, a common cause of seasonal allergic diseases affecting an increasing part of world population. Peptide shotgun experiments performed on nanoLiquid Ultra Pressure Chromatography coupled with fast Q-TOF MS-MS/MS acquisition protocols (MS(E)) and 2-DE immunoblot combined with MALDI-TOF-TOF analysis allowed the detection of all previously identified ryegrass allergens. Comparative analysis of immunoblot highlighted a class of patients characterized by a more complex 2-DE pattern associated with increased levels of IgE antibodies and by higher susceptibility to multiple sensitization toward different allergen sources. Cluster analysis revealed that all these patients recognized profilin, considered the main cross-reactive allergen in grass pollen. Furthermore, mass spectrometry analysis revealed the presence of other IgE reactive components in ryegrass pollen that might be involved in polysensitization, such as cyclophilin, fructosyltransferase and legumin-like protein.

Targeting phosphatase-dependent proteoglycan switch for rheumatoid arthritis therapy

Targeting joint-lining fibroblast-like synoviocytes reduces the severity of arthritis. Switching off arthritis In patients with rheumatoid arthritis (RA), joint-lining cells—fibroblast-like synoviocytes (FLS)—become activated and contribute to inflammation as well as cartilage and bone destruction. FLS express RPTPσ, which, in neurons, can be regulated by a proteoglycan switch. Doody et al. now find that FLS can also be regulated by this proteoglycan switch, and that an RPTPσ decoy protein can block this switch and decrease FLS invasiveness and severity of arthritis in human cells and a mouse model of RA. If these data hold true in humans, targeting this proteoglycan switch may add another option when treating patients with RA. Despite the availability of several therapies for rheumatoid arthritis (RA) that target the immune system, a large number of RA patients fail to achieve remission. Joint-lining cells, called fibroblast-like synoviocytes (FLS), become activated during RA and mediate joint inflammation and destruction of cartilage and bone. We identify RPTPσ, a transmembrane tyrosine phosphatase, as a therapeutic target for FLS-directed therapy. RPTPσ is reciprocally regulated by interactions with chondroitin sulfate or heparan sulfate containing extracellular proteoglycans in a mechanism called the proteoglycan switch. We show that the proteoglycan switch regulates FLS function. Incubation of FLS with a proteoglycan-binding RPTPσ decoy protein inhibited cell invasiveness and attachment to cartilage by disrupting a constitutive interaction between RPTPσ and the heparan sulfate proteoglycan syndecan-4. RPTPσ mediated the effect of proteoglycans on FLS signaling by regulating the phosphorylation and cytoskeletal localization of ezrin. Furthermore, administration of the RPTPσ decoy protein ameliorated in vivo human FLS invasiveness and arthritis severity in the K/BxN serum transfer model of RA. Our data demonstrate that FLS are regulated by an RPTPσ-dependent proteoglycan switch in vivo, which can be targeted for RA therapy. We envision that therapies targeting the proteoglycan switch or its intracellular pathway in FLS could be effective as a monotherapy or in combination with currently available immune-targeted agents to improve control of disease activity in RA patients.

PTP4A1 promotes TGFβ signaling and fibrosis in systemic sclerosis

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of skin and internal organs. Protein tyrosine phosphatases have received little attention in the study of SSc or fibrosis. Here, we show that the tyrosine phosphatase PTP4A1 is highly expressed in fibroblasts from patients with SSc. PTP4A1 and its close homolog PTP4A2 are critical promoters of TGFβ signaling in primary dermal fibroblasts and of bleomycin-induced fibrosis in vivo. PTP4A1 promotes TGFβ signaling in human fibroblasts through enhancement of ERK activity, which stimulates SMAD3 expression and nuclear translocation. Upstream from ERK, we show that PTP4A1 directly interacts with SRC and inhibits SRC basal activation independently of its phosphatase activity. Unexpectedly, PTP4A2 minimally interacts with SRC and does not promote the SRC–ERK–SMAD3 pathway. Thus, in addition to defining PTP4A1 as a molecule of interest for TGFβ-dependent fibrosis, our study provides information regarding the functional specificity of different members of the PTP4A subclass of phosphatases.Although protein tyrosine kinases are being explored as antifibrotic agents for the treatment of systemic sclerosis, little is known about the function of counteractive protein tyrosine phosphatases in this context. Here, the authors show that PTP4A1 is highly expressed by fibroblasts from patients with systemic sclerosis and promotes TGFβ activity via SRC–ERK–SMAD3 signaling.

Receptor Protein Tyrosine Phosphatase α-Mediated Enhancement of Rheumatoid Synovial Fibroblast Signaling and Promotion of Arthritis in Mice

During rheumatoid arthritis (RA), fibroblast‐like synoviocytes (FLS) critically promote disease pathogenesis by aggressively invading the extracellular matrix of the joint. The focal adhesion kinase (FAK) signaling pathway is emerging as a contributor to the anomalous behavior of RA FLS. The receptor protein tyrosine phosphatase α (RPTPα), which is encoded by the PTPRA gene, is a key promoter of FAK signaling. The aim of this study was to investigate whether RPTPα mediates FLS aggressiveness and RA pathogenesis.

TGFβ responsive tyrosine phosphatase promotes rheumatoid synovial fibroblast invasiveness

Objective In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) that line joint synovial membranes aggressively invade the extracellular matrix, destroying cartilage and bone. As signal transduction in FLS is mediated through multiple pathways involving protein tyrosine phosphorylation, we sought to identify protein tyrosine phosphatases (PTPs) regulating the invasiveness of RA FLS. We describe that the transmembrane receptor PTPκ (RPTPκ), encoded by the transforming growth factor (TGF) β-target gene, PTPRK, promotes RA FLS invasiveness. Methods Gene expression was quantified by quantitative PCR. PTP knockdown was achieved using antisense oligonucleotides. FLS invasion and migration were assessed in transwell or spot assays. FLS spreading was assessed by immunofluorescence microscopy. Activation of signalling pathways was analysed by Western blotting of FLS lysates using phosphospecific antibodies. In vivo FLS invasiveness was assessed by intradermal implantation of FLS into nude mice. The RPTPκ substrate was identified by pull-down assays. Results PTPRK expression was higher in FLS from patients with RA versus patients with osteoarthritis, resulting from increased TGFB1 expression in RA FLS. RPTPκ knockdown impaired RA FLS spreading, migration, invasiveness and responsiveness to platelet-derived growth factor, tumour necrosis factor and interleukin 1 stimulation. Furthermore, RPTPκ deficiency impaired the in vivo invasiveness of RA FLS. Molecular analysis revealed that RPTPκ promoted RA FLS migration by dephosphorylation of the inhibitory residue Y527 of SRC. Conclusions By regulating phosphorylation of SRC, RPTPκ promotes the pathogenic action of RA FLS, mediating cross-activation of growth factor and inflammatory cytokine signalling by TGFβ in RA FLS.

Receptor protein tyrosine phosphatase alpha enhances rheumatoid synovial fibroblast signaling and promotes arthritis in mice

During rheumatoid arthritis (RA), fibroblast‐like synoviocytes (FLS) critically promote disease pathogenesis by aggressively invading the extracellular matrix of the joint. The focal adhesion kinase (FAK) signaling pathway is emerging as a contributor to the anomalous behavior of RA FLS. The receptor protein tyrosine phosphatase α (RPTPα), which is encoded by the PTPRA gene, is a key promoter of FAK signaling. The aim of this study was to investigate whether RPTPα mediates FLS aggressiveness and RA pathogenesis.

Nitric Oxide Dependent Degradation of Polyethylene Glycol‐Modified Single‐Walled Carbon Nanotubes: Implications for Intra‐Articular Delivery

Polyethylene glycol (PEG)-modified carbon nanotubes have been successfully employed for intra-articular delivery in mice without systemic or local toxicity. However, the fate of the delivery system itself remains to be understood. In this study 2 kDa PEG-modified single-walled carbon nanotubes (PNTs) are synthesized, and trafficking and degradation following intra-articular injection into the knee-joint of healthy mice are studied. Using confocal Raman microspectroscopy, PNTs can be imaged in the knee-joint and are found to either egress from the synovial cavity or undergo biodegradation over a period of 3 weeks. Raman analysis discloses that PNTs are oxidatively degraded mainly in the chondrocyte-rich cartilage and meniscus regions while PNTs can also be detected in the synovial membrane regions, where macrophages can be found. Furthermore, using murine chondrocyte (ATDC-5) and macrophage (RAW264.7) cell lines, biodegradation of PNTs in activated, nitric oxide (NO)-producing chondrocytes, which is blocked upon pharmacological inhibition of inducible nitric oxide synthase (iNOS), can be shown. Biodegradation of PNTs in macrophages is also noted, but after a longer period of incubation. Finally, cell-free degradation of PNTs upon incubation with the peroxynitrite-generating compound, SIN-1 is demonstrated. The present study paves the way for the use of PNTs as delivery systems in the treatment of diseases of the joint.

Protein Tyrosine Phosphatases in Systemic Sclerosis: Potential Pathogenic Players and Therapeutic Targets

Purpose of ReviewThe pathogenesis of systemic sclerosis depends on a complex interplay between autoimmunity, vasculopathy, and fibrosis. Reversible phosphorylation on tyrosine residues, in response to growth factors and other stimuli, critically regulates each one of these three key pathogenic processes. Protein tyrosine kinases, the enzymes that catalyze addition of phosphate to tyrosine residues, are known players in systemic sclerosis, and tyrosine kinase inhibitors are undergoing clinical trials for treatment of this disease. Until recently, the role of tyrosine phosphatases—the enzymes that counteract the action of tyrosine kinases by removing phosphate from tyrosine residues—in systemic sclerosis has remained largely unknown. Here, we review the function of tyrosine phosphatases in pathways relevant to the pathogenesis of systemic sclerosis and their potential promise as therapeutic targets to halt progression of this debilitating rheumatic disease.Recent FindingsProtein tyrosine phosphatases are emerging as important regulators of a multitude of signaling pathways and undergoing validation as molecular targets for cancer and other common diseases. Recent advances in drug discovery are paving the ways to develop new classes of tyrosine phosphatase modulators to treat human diseases.SummaryAlthough so far only few reports have focused on tyrosine phosphatases in systemic sclerosis, these enzymes play a role in multiple pathways relevant to disease pathogenesis. Further studies in this field are warranted to explore the potential of tyrosine phosphatases as drug targets for systemic sclerosis.