Marianne Petro

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.

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.

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 11β-hydroxysteroid dehydrogenase-2 (11β-HSD2), which otherwise inactivates cortisol, sustaining tumor cortisol concentrations to stimulate GR and enzalutamide resistance. Remarkably, reinstatement of 11β-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. DOI: http://dx.doi.org/10.7554/eLife.20183.001

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.

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.

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.

The association of D4, 3-keto-abi (D4A) and response to abiraterone in castration-resistant prostate cancer (CRPC).

245 Background: Abiraterone (Abi), a potent inhibitor of 17α-hydroxylase/17,20-lyase (CYP17A1), is a standard treatment for men with metastatic CRPC. Abi is converted to D4A by 3β-hydroxysteroid dehydrogenase (3βHSD). D4A inhibits CYP17A1, 3βHSD, and steroid-5α-reductase (SRD5A) and has direct androgen receptor antagonist activity, which together make it a more potent agent than Abi in xenograft models. It is not known if conversion to D4A in patients (pts) correlates with response or resistance to Abi. Methods: Blood was collected (single time point on Abi) from CRPC pts who started Abi during 2011-2015. Abi and D4A were extracted from serum and analyzed by mass spectrometry. The purpose of this ongoing study is to assess the potential correlation between D4A and response to treatment. Results: 32 patients with CRPC had blood collected. 4 pts (12.5%) received ketoconazole and 6 (18.8%) chemotherapy prior to Abi. Median pre-Abi prostate-specific antigen (PSA) was 14.3 ng/ml (0.6-646.1). Median time from i...