Novel Chemokine-Based Immunotoxins for Potent and Selective Targeting of Cytomegalovirus Infected Cells

Immunotoxins as antiviral therapeutics are largely unexplored but have promising prospective due to their high selectivity potential and their unparalleled efficiency. One recent example targeted the virus-encoded G protein-coupled receptor US28 as a strategy for specific and efficient treatment of human cytomegalovirus (HCMV) infections. US28 is expressed on virus-infected cells and scavenge chemokines by rapid internalization. The chemokine-based fusion-toxin protein (FTP) consisted of a variant (F49A) of CX3CL1 specifically targeting US28 linked to the catalytic domain of Pseudomonas exotoxin A (PE). Here, we systematically seek to improve F49A-FTP by modifications in its three structural domains; we generated variants with (1) altered chemokine sequence (K14A, F49L, and F49E), (2) shortened and elongated linker region, and (3) modified toxin domain. Only F49L-FTP displayed higher selectivity in its binding to US28 versus CX3CR1, the endogenous receptor for CX3CL1, but this was not matched by a more selective killing of US28-expressing cells. A longer linker and different toxin variants decreased US28 affinity and selective killing. Thereby, F49A-FTP represents the best candidate for HCMV treatment. Many viruses encode internalizing receptors suggesting that not only HCMV but also, for instance, Epstein-Barr virus and Kaposi’s sarcoma-associated herpesvirus may be targeted by FTPs

Rationally designed chemokine-based toxin targeting the viral G protein-coupled receptor US28 potently inhibits cytomegalovirus infection in vivo

The use of receptor–ligand interactions to direct toxins to kill diseased cells selectively has shown considerable promise for treatment of a number of cancers and, more recently, autoimmune disease. Here we move the fusion toxin protein (FTP) technology beyond cancer/autoimmune therapeutics to target the human viral pathogen, human cytomegalovirus (HCMV), on the basis of its expression of the 7TM G protein-coupled chemokine receptor US28. The virus origin of US28 provides an exceptional chemokine-binding profile with high selectivity and improved binding for the CX3C chemokine, CX(3)CL1. Moreover, US28 is constitutively internalizing by nature, providing highly effective FTP delivery. We designed a synthetic CX(3)CL1 variant engineered to have ultra-high affinity for US28 and greater specificity for US28 than the natural sole receptor for CX(3)CL1, CX(3)CR1, and we fused the synthetic variant with the cytotoxic domain of Pseudomonas Exotoxin A. This novel strategy of a rationally designed FTP provided unparalleled anti-HCMV efficacy and potency in vitro and in vivo

Pathological tendon histology in early and chronic human patellar tendinopathy

The present pilot study investigated the extent of histological tissue changes in both chronic tendinopathy and in individuals that display early clinical signs of tendinopathy. The study included 8 individuals of whom 3 were healthy without any tendon symptoms, 2 had early symptoms (1–2 months), and 3 had chronic symptoms (>3 months) from their patellar tendons. Percutaneous needle biopsy samples were obtained from the affected tendon tissue region. Biopsy samples were stained with Haematoxylin & Eosin, and multiplex immunofluorescence staining was performed for markers of inflammation and resolution. Both early and chronic stage patellar tendon biopsy samples from this small patient cohort exhibited expansion of the interfascicular matrix (IFM) and endotenon regions together with increased cellularity and vascularity. These histological observations were moderate in early tendinopathy, whereas they were more pronounced and associated with marked disruption of tissue architecture in chronic tendinopathy. Early stage tendinopathic patellar tendons expressed markers associated with an activated phenotype of fibroblasts (CD90, CD34), macrophages (S100A8), and endothelial cells (ICAM1, VCAM1). These tissues also expressed enzymes implicated in inflammation (PTGS2, 15PGDH) and resolution (ALOX12) and the proresolving receptor ERV1. Immunopositive staining for these markers was predominantly located in the IFM regions. These preliminary findings suggest that mild to moderate structural histological changes including expansion of IFM and endotenon regions are pathological features of early tendinopathy, and support inflammatory and resolving processes are active in early-stage disease. Further investigation of the cellular and molecular basis of early-stage tendinopathy is required to inform therapeutic strategies that prevent the development of irreversible chronic tendon disease

Assessment of content validity and psychometric properties of VISA-A for Achilles tendinopathy.

A recent COSMIN review found that the Victorian Institute of Sports Assessment-Achilles tendinopathy questionnaire (VISA-A) has flawed construct validity. The objective of the current study was to assess specifically the process of how VISA-A was constructed and validated, and whether the Danish version of VISA-A is a valid patient-reported outcome measure (PROM) for measuring the perceived impact of Achilles tendinopathy. The original item generation strategy for content validity and the process for confirming the scaling properties (construct validity) were examined. In addition, construct validity was evaluated directly using several psychometric methods (Rasch analysis, confirmatory factor analysis (CFA), and multivariable linear regression) in a cohort of 318 persons with Achilles tendinopathy with symptom duration groups ranging from less than 3 months to more than 1 year of chronicity, and a group of 120 healthy persons. We found that the item generation and item reduction in the original construction of VISA-A was based on literature review and clinician consensus with little or no patient involvement. We determined that 1) VISA-A consists of ambiguous conceptual item themes and thus lacks content validity, 2) there was no thorough investigation of the psychometric properties of the original version of VISA-A, which thus lacks construct validity, and 3) rigorous direct assessment of the psychometric properties of the Danish VISA-A revealed inadequate psychometric properties. In agreement with the COSMIN study, we conclude that when used as a single score, VISA-A is not an adequate scale for measuring self-reported impact of Achilles tendinopathy