Mapping cellular senescence networks in human diabetic foot ulcers.

Cellular senescence, a cell fate defined by irreversible cell cycle arrest, has been observed to contribute to chronic age-related conditions including non-healing wounds, such as diabetic foot ulcers. However, the role of cellular senescence in the pathogenesis of diabetic foot ulcers remains unclear. To examine the contribution of senescent phenotypes to these chronic wounds, differential gene and network analyses were performed on publicly available bulk RNA sequencing of whole skin biopsies of wound edge diabetic foot ulcers and uninvolved diabetic foot skin. Wald tests with Benjamini-Hochberg correction were used to evaluate differential gene expression. Results showed that cellular senescence markers, CDKN1A, CXCL8, IGFBP2, IL1A, MMP10, SERPINE1, and TGFA, were upregulated, while TP53 was downregulated in diabetic foot ulcers compared to uninvolved diabetic foot skin. NetDecoder was then used to identify and compare context-specific protein-protein interaction networks using known cellular senescence markers as pathway sources. The diabetic foot ulcer protein-protein interaction network demonstrated significant perturbations with decreased inhibitory interactions and increased senescence markers compared to uninvolved diabetic foot skin. Indeed, TP53 (p53) and CDKN1A (p21) appeared to be key regulators in diabetic foot ulcer formation. These findings suggest that cellular senescence is an important mediator of diabetic foot ulcer pathogenesis.

Hybrid tumors with perineurioma components: a systematic review of the literature and illustrative case.

PURPOSE: Hybrid peripheral nerve sheath tumors (HPNST) are a newly recognized class of peripheral nerve sheath tumor, composed of at least two areas characteristic of perineurioma, schwannoma, or neurofibroma. The literature consists only of case reports and small series; therefore, we present an illustrative case and an analysis of all reported cases of HPNST with a perineurioma component in the literature. METHODS: A systematic search of the literature was performed to identify all reported cases of hybrid perineurioma-schwannoma or perineurioma-neurofibroma in the world's literature. Individual cases were analyzed for demographics, clinical features, imaging, and outcomes. RESULTS: A total of 159 cases were identified across 41 studies. Hybrid tumors tended to present in mid-adulthood (median 38.5 years), predominantly affected females (57%, 89/156), as a painless (63%, 63/100) mass, or swelling. Ten patients (10/74, 14%) had a history of neurofibromatosis 1, and 2 patients a history of neurofibromatosis 2 (2/74, 3%). The majority (78%, 122/157) of cases occurred superficially, most commonly in the lower extremity (25%, 39/157). Perineurioma-schwannoma was the most reported (86%, 137/159) pathologic diagnosis, with 3 cases presenting with malignant features. Two cases reocurred after resection. CONCLUSION: HPNST tend to occur in mid-adulthood and present as slowly progressive, painless, superficial masses, with a heterogeneous appearance on imaging. These entities pose a unique diagnostic challenge and likely remain under-recognized in the literature and current clinical practice. They pose low risk of recurrence or malignant transformation, and future work regarding the association with neurofibromatosis and genetic profiles is needed.

Strategies, considerations, and recent advancements in the development of liquid biopsy for glioblastoma: a step towards individualized medicine in glioblastoma.

OBJECTIVE: Glioblastoma (GBM) is a devasting primary brain tumor with less than a 5% 5-year survival. Treatment response assessment can be challenging because of inflammatory pseudoprogression that mimics true tumor progression clinically and on imaging. Developing additional noninvasive assays is critical. In this article, the authors review various biomarkers that could be used in developing liquid biopsies for GBM, along with strengths, limitations, and future applications. In addition, they present a potential liquid biopsy design based on the use of an extracellular vesicle-based liquid biopsy targeting nonneoplastic extracellular vesicles. METHODS: The authors conducted a current literature review of liquid biopsy in GBM by searching the PubMed, Scopus, and Google Scholar databases. Articles were assessed for type of biomarker, isolation methodology, analytical techniques, and clinical relevance. RESULTS: Recent work has shown that liquid biopsies of plasma, blood, and/or CSF hold promise as noninvasive clinical tools that can be used to diagnose recurrence, assess treatment response, and predict patient outcomes in GBM. Liquid biopsy in GBM has focused primarily on extracellular vesicles, cell-free tumor nucleic acids, and whole-cell isolates as focal biomarkers. GBM tumor signatures have been generated via analysis of tumor gene mutations, unique RNA expression, and metabolic and proteomic alterations. Liquid biopsies capture tumor heterogeneity, identifying alterations in GBM tumors that may be undetectable via surgical biopsy specimens. Finally, biomarker burden can be used to assess treatment response and recurrence in GBM. CONCLUSIONS: Liquid biopsy offers a promising avenue for monitoring treatment response and recurrence in GBM without invasive procedures. Although additional steps must be taken to bring liquid biopsy into the clinic, proof-of-principle studies and isolation methodologies are promising. Ultimately, CSF and/or plasma-based liquid biopsy is likely to be a powerful tool in the neurosurgeon's arsenal in the near future for the treatment and management of GBM patients.

Systems neuroimmunology: a review of multiomics methodologies to characterize neuroimmunological interactions in spinal and cranial diseases.

Neuroimmunology plays a critical role in our understanding of the pathophysiological processes that underlie a variety of diseases treated by neurosurgeons, including degenerative disc disease (DDD), glioblastoma (GBM), aneurysmal subarachnoid hemorrhage (aSAH), and others. Compared with traditional methods in neuroimmunology, which study one pathway or gene at a time, emerging multiomics methodologies allow for holistic interrogation of multiple immune-signaling pathways to test hypotheses and the effects of therapeutics at a systems level. In this review, the authors summarize key concepts for gathering and analyzing multiomics data so that neurosurgeons can contribute to the emerging field of systems neuroimmunology. Additionally, they describe 3 use cases, based on original research published by their group and others, that utilize transcriptomic, metabolomic, and proteomic analyses to study immune-signaling pathways in DDD, aSAH, and GBM. Through these use cases, techniques for performing machine learning and network-based analyses to generate new clinical insights from multiomics data are shared. The authors hope that neurosurgeons might use this review as a summary of common tools and principles in systems immunology to better engage in creating the immunotherapies of tomorrow.

NKG7 Is a T-cell-Intrinsic Therapeutic Target for Improving Antitumor Cytotoxicity and Cancer Immunotherapy.

Cytotoxic CD8+ T cells (CTL) are a crucial component of the immune system notable for their ability to eliminate rapidly proliferating malignant cells. However, the T-cell intrinsic factors required for human CTLs to accomplish highly efficient antitumor cytotoxicity are not well defined. By evaluating human CD8+ T cells from responders versus nonresponders to treatment with immune checkpoint inhibitors, we sought to identify key factors associated with effective CTL function. Single-cell RNA-sequencing analysis of peripheral CD8+ T cells from patients treated with anti-PD-1 therapy showed that cells from nonresponders exhibited decreased expression of the cytolytic granule-associated molecule natural killer cell granule protein-7 (NKG7). Functional assays revealed that reduced NKG7 expression altered cytolytic granule number, trafficking, and calcium release, resulting in decreased CD8+ T-cell-mediated killing of tumor cells. Transfection of T cells with NKG7 mRNA was sufficient to improve the tumor-cell killing ability of human T cells isolated from nonresponders and increase their response to anti-PD-1 or anti-PD-L1 therapy in vitro. NKG7 mRNA therapy also improved the antitumor activity of murine tumor antigen-specific CD8+ T cells in an in vivo model of adoptive cell therapy. Finally, we showed that the transcription factor ETS1 played a role in regulating NKG7 expression. Together, our results identify NKG7 as a necessary component for the cytotoxic function of CD8+ T cells and establish NKG7 as a T-cell-intrinsic therapeutic target for enhancing cancer immunotherapy.See related article by Li et al., p. 154.

Modular network mechanism of CCN1-associated resistance to HSV-1-derived oncolytic immunovirotherapies for glioblastomas.

Glioblastomas (GBMs) are the most common and lethal primary brain malignancy in adults. Oncolytic virus (OV) immunotherapies selectively kill GBM cells in a manner that elicits antitumor immunity. Cellular communication network factor 1 (CCN1), a protein found in most GBM microenvironments, expression predicts resistance to OVs, particularly herpes simplex virus type 1 (HSV-1). This study aims to understand how extracellular CCN1 alters the GBM intracellular state to confer OV resistance. Protein-protein interaction network information flow analyses of LN229 human GBM transcriptomes identified 39 novel nodes and 12 binary edges dominating flow in CCN1high cells versus controls. Virus response programs, notably against HSV-1, and cytokine-mediated signaling pathways are highly enriched. Our results suggest that CCN1high states exploit IDH1 and TP53, and increase dependency on RPL6, HUWE1, and COPS5. To validate, we reproduce our findings in 65 other GBM cell line (CCLE) and 174 clinical GBM patient sample (TCGA) datasets. We conclude through our generalized network modeling and system level analysis that CCN1 signals via several innate immune pathways in GBM to inhibit HSV-1 OVs before transduction. Interventions disrupting this network may overcome immunovirotherapy resistance.

Emerging workforce readiness in regenerative healthcare.

The biology of regenerative medicine has steadily matured, providing the foundation for randomized clinical trials and translation into validated applications. Today, the growing regenerative armamentarium is poised to impact disease management, yet a gap in training next-generation healthcare providers, equipped to adopt and deliver regenerative options, has been exposed. This special report highlights a multiyear experience in developing and deploying a comprehensive regenerative curriculum for medical trainees. For academicians and institutions invested in establishing a formalized regenerative medicine syllabus, the Regenerative Medicine and Surgery course provides a patient-focused prototype for next-generation learners, offering a dedicated educational experience that encompasses discovery, development and delivery of regenerative solutions. Built with the vision of an evolving regenerative care model, this transdisciplinary endeavor could serve as an adoptable education portal to advance the readiness of the emergent regenerative healthcare workforce globally.

Synthetic and systems biology principles in the design of programmable oncolytic virus immunotherapies for glioblastoma.

Oncolytic viruses (OVs) are a class of immunotherapeutic agents with promising preclinical results for the treatment of glioblastoma (GBM) but have shown limited success in recent clinical trials. Advanced bioengineering principles from disciplines such as synthetic and systems biology are needed to overcome the current challenges faced in developing effective OV-based immunotherapies for GBMs, including off-target effects and poor clinical responses. Synthetic biology is an emerging field that focuses on the development of synthetic DNA constructs that encode networks of genes and proteins (synthetic genetic circuits) to perform novel functions, whereas systems biology is an analytical framework that enables the study of complex interactions between host pathways and these synthetic genetic circuits. In this review, the authors summarize synthetic and systems biology concepts for developing programmable, logic-based OVs to treat GBMs. Programmable OVs can increase selectivity for tumor cells and enhance the local immunological response using synthetic genetic circuits. The authors discuss key principles for developing programmable OV-based immunotherapies, including how to 1) select an appropriate chassis, a vector that carries a synthetic genetic circuit, and 2) design a synthetic genetic circuit that can be programmed to sense key signals in the GBM microenvironment and trigger release of a therapeutic payload. To illustrate these principles, some original laboratory data are included, highlighting the need for systems biology studies, as well as some preliminary network analyses in preparation for synthetic biology applications. Examples from the literature of state-of-the-art synthetic genetic circuits that can be packaged into leading candidate OV chassis are also surveyed and discussed.

Oncolytic virus-derived type I interferon restricts CAR T cell therapy.

The application of adoptive T cell therapies, including those using chimeric antigen receptor (CAR)-modified T cells, to solid tumors requires combinatorial strategies to overcome immune suppression associated with the tumor microenvironment. Here we test whether the inflammatory nature of oncolytic viruses and their ability to remodel the tumor microenvironment may help to recruit and potentiate the functionality of CAR T cells. Contrary to our hypothesis, VSVmIFNß infection is associated with attrition of murine EGFRvIII CAR T cells in a B16EGFRvIII model, despite inducing a robust proinflammatory shift in the chemokine profile. Mechanistically, type I interferon (IFN) expressed following infection promotes apoptosis, activation, and inhibitory receptor expression, and interferon-insensitive CAR T cells enable combinatorial therapy with VSVmIFNß. Our study uncovers an unexpected mechanism of therapeutic interference, and prompts further investigation into the interaction between CAR T cells and oncolytic viruses to optimize combination therapy.

Venous thromboembolic events in patients undergoing craniotomy for tumor resection: incidence, predictors, and review of literature.

OBJECTIVE: The authors sought to investigate the incidence and predictors of venous thromboembolic events (VTEs) after craniotomy for tumor resection, which are not well established, and the efficacy of and risks associated with VTE chemoprophylaxis, which remains controversial. METHODS: The authors investigated the incidence of VTEs in a consecutive series of patients presenting to the authors' institution for resection of an intracranial lesion between 2012 and 2017. Information on patient and tumor characteristics was collected and independent predictors of VTEs were determined using stepwise multivariate logistic regression analysis. Review of the literature was performed by searching MEDLINE using the keywords "venous thromboembolism," "deep venous thrombosis," "pulmonary embolism," "craniotomy," and "brain neoplasms." RESULTS: There were 1622 patients included for analysis. A small majority of patients were female (52.6%) and the mean age of the cohort was 52.9 years (SD 15.8 years). A majority of intracranial lesions were intraaxial (59.3%). The incidence of VTEs was 3.0% and the rates of deep venous thromboses and pulmonary emboli were 2.3% and 0.9%, respectively. On multivariate analysis, increasing patient age (unit OR 1.02, 95% CI 1.00-1.05; p = 0.018), history of VTE (OR 7.26, 95% CI 3.24-16.27; p < 0.001), presence of motor deficit (OR 2.64, 95% CI 1.43-4.88; p = 0.002), postoperative intracranial hemorrhage (OR 4.35, 95% CI 1.51-12.55; p < 0.001), and prolonged intubation or reintubation (OR 3.27, 95% CI 1.28-8.32; p < 0.001) were independently associated with increased odds of a VTE. There were 192 patients who received VTE chemoprophylaxis (11.8%); the mean postoperative day of chemoprophylaxis initiation was 4.6 (SD 3.8). The incidence of VTEs was higher in patients receiving chemoprophylaxis than in patients not receiving chemoprophylaxis (8.3% vs 2.2%; p < 0.001). There were 30 instances of clinically significant postoperative hemorrhage (1.9%), with only 1 hemorrhage occurring after initiation of VTE chemoprophylaxis (0.1%). CONCLUSIONS: The study results show the incidence and predictors of VTEs after craniotomy for tumor resection in this patient population. The incidence of VTE within this cohort appears low and comparable to that observed in other institutional series, despite the lack of routine prophylactic anticoagulation in the postoperative setting.

Pathogenesis of thrombosis: cellular and pharmacogenetic contributions.

Our understanding of thrombosis formation has evolved significantly ever since physician Rudolf Virchow proposed his "triad" theory in 1856. Modern science has elucidated the mechanisms of stasis, hypercoagulability, and endothelial dysfunction. Today, we have a firm understanding of the key molecular factors involved in the coagulation cascade and fibrinolytic system, as well as the underlying genetic influences. This knowledge of cellular and genetic contributors has been translated into diverse pharmaceutical interventions. Here, we examine the molecular and cellular mechanisms of thrombosis and its associated pathologies. We also review the current state of pharmacologic interventions, including pro- and anti-thrombotics, direct oral anticoagulants, and anti-platelet therapies. The pharmacogenetic factors that guide clinical decision making and prognosis are described in detail. Finally, we explore new approaches to thrombosis drug discovery, repurposing, and diagnostics. We argue that network biology tools will enable a systems pharmacology revolution in the next generation of interventions, facilitating precision medicine applications and ultimately leading to improved patient outcomes.

Inhibition of immune activation by a novel nuclear factor-kappa B inhibitor in HTLV-I-associated neurologic disease.

The human T-lymphotropic virus type I (HTLV-I) causes a chronic inflammatory disorder of the central nervous system termed HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-I encodes a protein known to activate several host-signaling pathways involved in inflammation, such as the nuclear factor-κB (NF-κB). The contribution of the NF-κB pathway to the pathogenesis of HAM/TSP, however, has not been fully defined. We show evidence of canonical NF-κB activation in short-term cultures of peripheral blood mononuclear cells (PBMCs) from subjects with HAM/TSP. NF-κB activation was closely linked to HTLV-I viral protein expression. The NF-κB activation in HAM/TSP PBMCs was reversed by a novel small-molecule inhibitor that demonstrates potent and selective NF-κB antagonist activity. Inhibition of NF-κB activation led to a reduction in the expression of lymphocyte activation markers and resulted in reduced cytokine signaling in HAM/TSP PBMCs. Furthermore, NF-κB inhibition led to a reduction in spontaneous lymphoproliferation, a key ex vivo correlate of the immune activation associated with HAM/TSP. These results indicate that NF-κB activation plays a critical upstream role in the immune activation of HAM/TSP, and identify the NF-κB pathway as a potential target for immunomodulation in HAM/TSP.

Measuring the activity of BioBrick promoters using an in vivo reference standard.

BACKGROUND: The engineering of many-component, synthetic biological systems is being made easier by the development of collections of reusable, standard biological parts. However, the complexity of biology makes it difficult to predict the extent to which such efforts will succeed. As a first practical example, the Registry of Standard Biological Parts started at MIT now maintains and distributes thousands of BioBrick standard biological parts. However, BioBrick parts are only standardized in terms of how individual parts are physically assembled into multi-component systems, and most parts remain uncharacterized. Standardized tools, techniques, and units of measurement are needed to facilitate the characterization and reuse of parts by independent researchers across many laboratories. RESULTS: We found that the absolute activity of BioBrick promoters varies across experimental conditions and measurement instruments. We choose one promoter (BBa_J23101) to serve as an in vivo reference standard for promoter activity. We demonstrated that, by measuring the activity of promoters relative to BBa_J23101, we could reduce variation in reported promoter activity due to differences in test conditions and measurement instruments by approximately 50%. We defined a Relative Promoter Unit (RPU) in order to report promoter characterization data in compatible units and developed a measurement kit so that researchers might more easily adopt RPU as a standard unit for reporting promoter activity. We distributed a set of test promoters to multiple labs and found good agreement in the reported relative activities of promoters so measured. We also characterized the relative activities of a reference collection of BioBrick promoters in order to further support adoption of RPU-based measurement standards. CONCLUSION: Relative activity measurements based on an in vivoreference standard enables improved measurement of promoter activity given variation in measurement conditions and instruments. These improvements are sufficient to begin to support the measurement of promoter activities across many laboratories. Additional in vivo reference standards for other types of biological functions would seem likely to have similar utility, and could thus improve research on the design, production, and reuse of standard biological parts.