A novel method to produce synthetic murine CXCL10 for efficient screening of functional variants.

Antitumor immune responses depend on the infiltration of solid tumors by effector T cells, a process guided by chemokines. In particular, the chemokine CXCL10 has been shown to play a critical role in mediating recruitment of CXCR3 + cytolytic T and NK cells in tumors, though its use as a therapeutic agent has not been widely explored. One of the limitations is due to the rapid inactivation of CXCL10 by dipeptidyl peptidase 4 (DPP4), a broadly expressed enzyme that is active in plasma and other bodily fluids. In the present study, we describe a novel method to produce synthetic CXCL10 that is resistant to DPP4 N-terminal truncation. Using a Fmoc solid-phase peptide synthesis approach, synthetic murine WT CXCL10 was produced, showing similar biochemical and biological properties to the recombinant protein. This synthesis method supported production of natural (amino acid substitution, insertion or deletion) and non-natural (chemical modifications) variants of CXCL10. In association with a functional screening cascade that assessed DPP4-mediated cleavage, CXCR3 signaling potency and chemotactic activity, we successfully generated 20 murine CXCL10 variants. Among those, two non-natural variants with N-methylated Leu3 (MeLeu3) and a reduced amide bond between Pro2 and Leu3 (rLeu3), respectively, showed resistance to DPP4 truncation but decreased CXCR3 signaling and chemotactic activity. Interestingly, MeLeu3 and rLeu3 CXCL10 behaved as DPP4 inhibitors, preventing the truncation of WT CXCL10. This study highlights the potential of using Fmoc solid-phase chemistry in association with biochemical and biological characterization to rapidly identify CXCL10 variants with desired properties. These novel methods unlock the opportunity to develop DPP4 resistant CXCL10 variants, as well as other chemokine substrates, while maintaining chemotactic properties.

Clinical and histopathologic features of paraneoplastic granuloma annulare in association with solid organ malignancies: A case-control study.

BACKGROUND: Granuloma annulare (GA) is a granulomatous skin eruption rarely associated with cancer. We report seven cases of paraneoplastic GA in association with solid organ malignancy. OBJECTIVE: To compare the clinical and histopathological features of paraneoplastic GA to case-matched controls of classic GA. METHODS: Retrospective chart and histopathological review of 7 individuals and 13 age- and sex-matched controls. Paraneoplastic GA was defined as GA occurring within 6 months of the diagnosis of solid organ malignancy and/or persistent GA that resolved with cancer treatment. RESULTS: Most cases of paraneoplastic GA were associated with lung cancer (4/7). The clinical and histopathological features of paraneoplastic and classic GA were similar. Compared to classic GA, paraneoplastic GA cases were more often generalized disease (6/7 vs 6/13), refractory to treatment, and had a perivascular inflammatory cell infiltrate (5/7 vs 2/13). All cases of paraneoplastic GA that underwent definitive treatment of their cancer improved. LIMITATIONS: Single-institution, retrospective review with a small sample size. CONCLUSION: Paraneoplastic GA is rare, similar to classic GA, and refractory to treatment. We advocate for age-appropriate screening in individuals with GA that is nonresponsive to multiple lines of systemic treatment and evaluating patients with concerning signs or symptoms for an underlying neoplasm.

Melanoma mimicking Rosai-Dorfman disease.

Despite well-defined clinical and histopathological features of melanoma, atypical presentations mimicking other skin disorders can result in a delayed diagnosis or misdiagnosis and subsequent inappropriate treatment. Rosai-Dorfman disease (RDD) is a rare histiocytic disorder with unique clinical and histopathological features. We report a case of melanoma treated with cryotherapy that mimicked RDD both clinically and histopathologically. We compare this RDD-like melanoma to classic RDD, outlining the importance of clinicopathological correlation prior to treatment, as well as the potential pitfalls in diagnosis after cryotherapy of pigmented lesions.

A Non-immunogenic Bivalent d-Protein Potently Inhibits Retinal Vascularization and Tumor Growth.

We report a general approach to engineering multivalent d-proteins with antibody-like activities in vivo. Mirror-image phage display and structure-guided design were utilized to create a d-protein that uses receptor mimicry to antagonize vascular endothelial growth factor A (VEGF-A). Selections against the d-protein form of VEGF-A using phage-displayed libraries of two different domain scaffolds yielded two proteins that bound distinct receptor interaction sites on VEGF-A. X-ray crystal structures of the d-protein/VEGF-A complexes were used to guide affinity maturation and to construct a heterodimeric d-protein VEGF-A antagonist with picomolar activity. The d-protein VEGF-A antagonist prevented vascular leakage in a rabbit eye model of wet age-related macular degeneration and slowed tumor growth in the MC38 syngeneic mouse tumor model with efficacies comparable to those of approved antibody drugs, and in contrast with antibodies, the d-protein was non-immunogenic during treatment and following subcutaneous immunizations.

Polymeric biomaterials for biophotonic applications.

With the growing importance of optical techniques in medical diagnosis and treatment, there exists a pressing need to develop and optimize materials platform for biophotonic applications. Particularly, the design of biocompatible and biodegradable materials with desired optical, mechanical, chemical, and biological properties is required to enable clinically relevant biophotonic devices for translating in vitro optical techniques into in situ and in vivo use. This technological trend propels the development of natural and synthetic polymeric biomaterials to replace traditional brittle, nondegradable silica glass based optical materials. In this review, we present an overview of the advances in polymeric optical material development, optical device design and fabrication techniques, and the accompanying applications to imaging, sensing and phototherapy.

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix.

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis(1). Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.