Catalytic antioxidant nanoparticles mitigate secondary injury progression and promote functional recovery in spinal cord injury model.

Traumatic spinal cord injury exacerbates disability with time due to secondary injury cascade triggered largely by overproduction of reactive oxygen species (ROS) at the lesion site, causing oxidative stress. This study explored nanoparticles containing antioxidant enzymes (antioxidant NPs) to neutralize excess ROS at the lesion site and its impact. When tested in a rat contusion model of spinal cord injury, a single dose of antioxidant NPs, administered intravenously three hours after injury, effectively restored the redox balance at the lesion site, interrupting the secondary injury progression. This led to reduced spinal cord tissue inflammation, apoptosis, cavitation, and inhibition of syringomyelia. Moreover, the treatment reduced scar tissue forming collagen at the lesion site, protected axons from demyelination, and stimulated lesion healing, with further analysis indicating the formation of immature neurons. The ultimate effect of the treatment was improved motor and sensory functions and rapid post-injury weight loss recovery. Histological analysis revealed activated microglia in the spinal cord displaying rod-shaped anti-inflammatory and regenerative phenotype in treated animals, contrasting with amoeboid inflammatory and degenerative phenotype in untreated control. Overall data suggest that restoring redox balance at the lesion site shifts the dynamics in the injured spinal cord microenvironment from degenerative to regenerative, potentially by promoting endogenous repair mechanisms. Antioxidant NPs show promise to be developed as an early therapeutic intervention in stabilizing injured spinal cord for enhanced recovery.

A Novel Method Using Fine Needle Aspiration from Tumor-Draining Lymph Nodes Could Enable the Discovery of New Prognostic Markers in Patients with Cutaneous Squamous Cell Carcinoma.

Cutaneous squamous cell cancer (cSCC) is the second most common form of skin cancer, characterized by abnormal, accelerated growth of squamous cells. When caught early, most cSCCs are curable. About 5 percent of the cSCC cases have advanced to such an extent, generally metastatic, that they are far more dangerous, with very poor prognosis and challenging to treat. All efforts to find biomarkers, in blood or in the tumor itself, for early identification of patients with a risk for metastasis have so far failed. The present study describes a novel method that enables the identification of lymphocyte markers in tumor-draining lymph nodes. Six patients with advanced cSCC were analyzed using a combination of a sentinel lymph node biopsy (SLNB) protocol, fine needle aspiration (FNA), and flow cytometry. Immunological results from the sentinel nodes were combined with corresponding data from peripheral blood and unfixed tumor tissues. The result demonstrates a striking difference between the subsets of T-cells from the three compartments. Our interpretation of this first pilot study is that the ability to follow specific immunological markers on lymphocytes in tumor-draining lymph nodes will enable the identification of novel prognostic biomarkers not detectable in material from blood and tumor tissues.

Upregulated expression of Notch1/4 - JAG-1/DLL-1 detected in allergic rhinitis.

BACKGROUND: Allergic rhinitis (AR) is a chronic disease with high prevalence. There are currently many treatments available. However, despite an often good therapeutic response, many patients still report impairment in quality of life (QoL) during the pollen season. A skewed T helper (Th)2 polarization is a well-acknowledged pathologic feature of AR. In animal models, local notch signaling in peripheral tissue seems crucial for Th2 cell differentiation and the development of AR. However, the involvement of Notch signaling in Th2 cell differentiation and the development of AR in humans remains unknown. Hence, the present study investigated the human expression of Notch receptors on CD4+ T-cells in nasal mucosa and blood. Correspondingly Notch ligand expression was assessed on nasal epithelial cells and neutrophils. METHODOLOGY: Nasal brush and blood samples from 18 patients with pollen-induced AR and 22 healthy controls were collected outside the pollen season. Notch 1-4 and Jagged-1,2 and Delta-like ligand 1,3-4 was analyzed using flow cytometry. RESULTS: The fraction of CD4+Notch1+ and CD4+Notch4+ T-cells was higher in AR patients than in healthy control patients. Further, the expression levels of the Notch ligands JAG-1 and DLL-1 were increased in nasal epithelial cells from AR patients compared to healthy control patients. In addition, AR patients displayed higher expression of JAG-1 on neutrophils both in the nasal mucosa and in peripheral blood. CONCLUSION: The present study is the first to demonstrate increased activity in the Notch1/4 - JAG-1/DLL-1 pathways among allergic individuals. Further propagating the importance of Notch signalling in AR and blocking JAG-1 and DLL-1-induced Notch signalling by nasal epithelial cells and Neutrophils are potential targets to reduce allergic airway inflammation.

Increased IL-26 associates with markers of hyperinflammation and tissue damage in patients with acute COVID-19.

Interleukin-26 (IL-26) is released by several immune and structural cells following stimulation of toll-like receptors (TLRs), whereupon it can directly inhibit viral replication and enhance neutrophil chemotaxis. Given these unique properties, IL-26 has emerged as an intriguing mediator of host defense in the lungs. However, the role of IL-26 in COVID-19 has not been thoroughly investigated. Here, we characterized the involvement of IL-26 in the hyperinflammation and tissue damage that occurs in patients with acute COVID-19. We found that IL-26 is markedly increased in blood samples from these patients, and that the concentration of IL-26 correlates with those of the neutrophil-mobilizing cytokines IL-8 and TNFα, respectively. Moreover, the increase in blood IL-26 correlates with enhanced surface expression of the "don't eat me" signal CD47 on blood neutrophils isolated from patients with acute COVID-19. Finally, we found that the blood concentration of IL-26 correlates with that of increased lactate dehydrogenase, an established marker of tissue damage, and decreased mean corpuscular hemoglobin (MCH), a previously verified hematological aberration in COVID-19, both of which are associated with severe disease. Thus, our findings indicate that increased systemic IL-26 associates with markers of hyperinflammation and tissue damage in patients with acute COVID-19, thereby forwarding the kinocidin IL-26 as a potential target for diagnosis, monitoring, and therapy in this deadly disease.

A prolonged innate systemic immune response in COVID-19.

Despite the introduction of vaccines, COVID-19 still affects millions of people worldwide. A better understanding of pathophysiology and the discovery of novel therapies are needed. One of the cells of interest in COVID-19 is the neutrophil. This cell type is being recruited to a site of inflammation as one of the first immune cells. In this project, we investigated a variety of neutrophils phenotypes during COVID-19 by measuring the expression of markers for migration, maturity, activation, gelatinase granules and secondary granules using flow cytometry. We show that neutrophils during COVID-19 exhibit altered phenotypes compared to healthy individuals. The activation level including NETs production and maturity of neutrophils seem to last longer during COVID-19 than expected for innate immunity. Neutrophils as one of the drivers of severe cases of COVID-19 are considered as potential treatment targets. However, for a successful implementation of treatment, there is a need for a better understanding of neutrophil functions and phenotypes in COVID-19. Our study answers some of those questions.

Loss of dihydrotestosterone-inactivation activity promotes prostate cancer castration resistance detectable by functional imaging.

Androgens such as testosterone and dihydrotestosterone are a critical driver of prostate cancer progression. Cancer resistance to androgen deprivation therapies ensues when tumors engage metabolic processes that produce sustained androgen levels in the tissue. However, the molecular mechanisms involved in this resistance process are unclear, and functional imaging modalities that predict impending resistance are lacking. Here, using the human LNCaP and C4-2 cell line models of prostate cancer, we show that castration treatment-sensitive prostate cancer cells that normally have an intact glucuronidation pathway that rapidly conjugates and inactivates dihydrotestosterone and thereby limits androgen signaling, become glucuronidation deficient and resistant to androgen deprivation. Mechanistically, using CRISPR/Cas9-mediated gene ablation, we found that loss of UDP glucuronosyltransferase family 2 member B15 (UGT2B15) and UGT2B17 is sufficient to restore free dihydrotestosterone, sustained androgen signaling, and development of castration resistance. Furthermore, loss of glucuronidation enzymatic activity was also detectable with a nonsteroid glucuronidation substrate. Of note, glucuronidation-incompetent cells and the resultant loss of intracellular conjugated dihydrotestosterone were detectable in vivo by 18F-dihydrotestosterone PET. Together, these findings couple a mechanism with a functional imaging modality to identify impending castration resistance in prostate cancers.

Aberrant corticosteroid metabolism in tumor cells enables GR takeover in enzalutamide resistant prostate cancer.

Prostate cancer is driven by androgen stimulation of the androgen receptor (AR). The next-generation AR antagonist, enzalutamide, prolongs survival, but resistance and lethal disease eventually prevail. Emerging data suggest that the glucocorticoid receptor (GR) is upregulated in this context, stimulating expression of AR-target genes that permit continued growth despite AR blockade. However, countering this mechanism by administration of GR antagonists is problematic because GR is essential for life. We show that enzalutamide treatment in human models of prostate cancer and patient tissues is accompanied by a ubiquitin E3-ligase, AMFR, mediating loss of 11ß-hydroxysteroid dehydrogenase-2 (11ß-HSD2), which otherwise inactivates cortisol, sustaining tumor cortisol concentrations to stimulate GR and enzalutamide resistance. Remarkably, reinstatement of 11ß-HSD2 expression, or AMFR loss, reverses enzalutamide resistance in mouse xenograft tumors. Together, these findings reveal a surprising metabolic mechanism of enzalutamide resistance that may be targeted with a strategy that circumvents a requirement for systemic GR ablation.

Tissue plasminogen activator followed by antioxidant-loaded nanoparticle delivery promotes activation/mobilization of progenitor cells in infarcted rat brain.

Inherent neuronal and circulating progenitor cells play important roles in facilitating neuronal and functional recovery post stroke. However, this endogenous repair process is rather limited, primarily due to unfavorable conditions in the infarcted brain involving reactive oxygen species (ROS)-mediated oxidative stress and inflammation following ischemia/reperfusion injury. We hypothesized that during reperfusion, effective delivery of antioxidants to ischemic brain would create an environment without such oxidative stress and inflammation, thus promoting activation and mobilization of progenitor cells in the infarcted brain. We administered recombinant human tissue-type plasminogen activator (tPA) via carotid artery at 3 h post stroke in a thromboembolic rat model, followed by sequential administration of the antioxidants catalase (CAT) and superoxide dismutase (SOD), encapsulated in biodegradable nanoparticles (nano-CAT/SOD). Brains were harvested at 48 h post stroke for immunohistochemical analysis. Ipsilateral brain slices from animals that had received tPA + nano-CAT/SOD showed a widespread distribution of glial fibrillary acidic protein-positive cells (with morphology resembling radial glia-like neural precursor cells) and nestin-positive cells (indicating the presence of immature neurons); such cells were considerably fewer in untreated animals or those treated with tPA alone. Brain sections from animals receiving tPA + nano-CAT/SOD also showed much greater numbers of SOX2- and nestin-positive progenitor cells migrating from subventricular zone of the lateral ventricle and entering the rostral migratory stream than in t-PA alone treated group or untreated control. Further, animals treated with tPA + nano-CAT/SOD showed far fewer caspase-positive cells and fewer neutrophils than did other groups, as well as an inhibition of hippocampal swelling. These results suggest that the antioxidants mitigated the inflammatory response, protected neuronal cells from undergoing apoptosis, and inhibited edema formation by protecting the blood-brain barrier from ROS-mediated reperfusion injury. A longer-term study would enable us to determine if our approach would assist progenitor cells to undergo neurogenesis and to facilitate neurological and functional recovery following stroke and reperfusion injury.

Blast-Associated Shock Waves Result in Increased Brain Vascular Leakage and Elevated ROS Levels in a Rat Model of Traumatic Brain Injury.

Blast-associated shock wave-induced traumatic brain injury (bTBI) remains a persistent risk for armed forces worldwide, yet its detailed pathophysiology remains to be fully investigated. In this study, we have designed and characterized a laboratory-scale shock tube to develop a rodent model of bTBI. Our blast tube, driven by a mixture of oxygen and acetylene, effectively generates blast overpressures of 20-130 psi, with pressure-time profiles similar to those of free-field blast waves. We tested our shock tube for brain injury response to various blast wave conditions in rats. The results show that blast waves cause diffuse vascular brain damage, as determined using a sensitive optical imaging method based on the fluorescence signal of Evans Blue dye extravasation developed in our laboratory. Vascular leakage increased with increasing blast overpressures and mapping of the brain slices for optical signal intensity indicated nonhomogeneous damage to the cerebral vasculature. We confirmed vascular leakage due to disruption in the blood-brain barrier (BBB) integrity following blast exposure. Reactive oxygen species (ROS) levels in the brain also increased with increasing blast pressures and with time post-blast wave exposure. Immunohistochemical analysis of the brain sections analyzed at different time points post blast exposure demonstrated astrocytosis and cell apoptosis, confirming sustained neuronal injury response. The main advantages of our shock-tube design are minimal jet effect and no requirement for specialized equipment or facilities, and effectively generate blast-associated shock waves that are relevant to battle-field conditions. Overall data suggest that increased oxidative stress and BBB disruption could be the crucial factors in the propagation and spread of neuronal degeneration following blast injury. Further studies are required to determine the interplay between increased ROS activity and BBB disruption to develop effective therapeutic strategies that can prevent the resulting cascade of neurodegeneration.

Cutaneous tumors cease CXCL9/Mig production as a result of IFN-γ-mediated immunoediting.

During growth in the host, tumor cells are subjected to the stresses of innate and adaptive immunity (immunoediting), which provoke epigenetic changes in the tumor and increase tumor resistance to these immune responses. Our recent studies in methylcholanthrene-induced fibrosarcomas have indicated the appearance and rapid growth of tumor variants deficient in producing the T cell chemoattractant chemokine CXCL9/Mig, an important component of antitumor immunity. In the current report, we demonstrate that highly tumorigenic Mig-deficient tumor variants arise in both cutaneous fibrosarcoma and melanoma as a result of immune stress imposed by IFN-γ and T cells. The consequence of the loss of tumor-derived Mig expression is the increased resistance of Mig-deficient tumors to T cell-mediated immunity, which promotes the accelerated growth of these tumor variants. Remarkably, the ability of Mig-deficient tumor cells to express another CXCR3 ligand, CXCL10/IFN-γ-inducible protein, does not compensate for the absent antitumor functions of Mig, suggesting a nonredundant role for this chemokine in the suppression of tumor growth. To our knowledge, these studies report for the first time that IFN-γ-mediated stress leads to the loss of specific chemokine expression by tumor cells, which in turn promotes tumor growth and evasion of the immune response.

A TLR5 agonist inhibits acute renal ischemic failure.

Reperfusion of ischemic organs induces a potent inflammatory response initiated by the generation of reactive oxygen species that directly damage tissue and promote leukocyte infiltration and activation that also mediate tissue injury. We recently found that radiation-induced tissue injury, which is caused by radiation-induced reactive oxygen species, is attenuated by administration of CBLB502, a pharmacologically optimized derivative of the TLR5 agonist flagellin. Therefore, we tested the ability of CBLB502 to attenuate injury in a murine model of acute ischemic renal failure. CBLB502 given 30 min before imposition of bilateral renal pedicle occlusion provided marked protection against the renal dysfunction and inflammation that follows reperfusion of ischemic kidneys, including marked decreases in leukocyte infiltration, proinflammatory cytokine production, and tubular injury. Importantly, CBLB502 given within 30 min after ischemic kidney reperfusion reproduced the protective effects of pretreatment with the TLR5 agonist, indicating a window following reperfusion in which CBLB502 administration abrogates acute renal ischemic failure. Bone marrow-reconstituted chimeras were used to show that the protective effects of CBLB502 could be delivered by intact MyD88 signaling on renal parenchymal cells. Consistent with this, Ab staining of kidney sections indicated that cells lining the renal vasculature expressed TLR5. Overall, these results indicate the use of TLR5 agonists as mitigators and protectants of acute renal ischemic failure.

High renal ischemia temperature increases neutrophil chemoattractant production and tissue injury during reperfusion without an identifiable role for CD4 T cells in the injury.

Various leukocyte populations, including neutrophils and CD4 T cells, have been implicated as mediators of acute renal ischemic injury. The influence of ischemic temperature on molecular and cellular mechanisms mediating this injury was tested in a mouse model. Wild-type C57BL/6, B6.CD4(-/-), B6.CD8(-/-), and B6.RAG-1(-/-) mice subjected to bilateral renal pedicle occlusion for 30 min at a higher (37 degrees C) but not a lower (32 degrees C) ischemic maintenance temperature had clear evidence of renal dysfunction and histopathology. Ischemia imposed at the higher temperature also increased CXCL1/KC and CXCL2/MIP-2 levels and neutrophils, but not T cells or macrophages, infiltrating into the ischemic kidneys. Depletion of neutrophils but not T cells attenuated the acute ischemic injury. These results indicate the influence of ischemic temperature and time on the production of neutrophil chemoattractants and subsequent neutrophil infiltration to mediate acute ischemic injury but fail to identify a role for adaptive immune components in this injury.