Erdafitinib in BCG-treated high-risk non-muscle-invasive bladder cancer.

BACKGROUND: Treatment options are limited for patients with high-risk non-muscle-invasive bladder cancer (NMIBC) with disease recurrence after bacillus Calmette-Guérin (BCG) treatment and who are ineligible for/refuse radical cystectomy. FGFR alterations are commonly detected in NMIBC. We evaluated the activity of oral erdafitinib, a selective pan-fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitor, versus intravesical chemotherapy in patients with high-risk NMIBC and select FGFR3/2 alterations following recurrence after BCG treatment. PATIENTS AND METHODS: Patients aged ≥18 years with recurrent, BCG-treated, papillary-only high-risk NMIBC (high-grade Ta/T1) and select FGFR alterations refusing or ineligible for radical cystectomy were randomized to 6 mg daily oral erdafitinib or investigator's choice of intravesical chemotherapy (mitomycin C or gemcitabine). The primary endpoint was recurrence-free survival (RFS). The key secondary endpoint was safety. RESULTS: Study enrollment was discontinued due to slow accrual. Seventy-three patients were randomized 2 : 1 to erdafitinib (n = 49) and chemotherapy (n = 24). Median follow-up for RFS was 13.4 months for both groups. Median RFS was not reached for erdafitinib [95% confidence interval (CI) 16.9 months-not estimable] and was 11.6 months (95% CI 6.4-20.1 months) for chemotherapy, with an estimated hazard ratio of 0.28 (95% CI 0.1-0.6; nominal P value = 0.0008). In this population, safety results were generally consistent with known profiles for erdafitinib and chemotherapy. CONCLUSIONS: Erdafitinib prolonged RFS compared with intravesical chemotherapy in patients with papillary-only, high-risk NMIBC harboring FGFR alterations who had disease recurrence after BCG therapy and refused or were ineligible for radical cystectomy.

Improving polarized neutron imaging for visualization of the Meissner effect in superconductors.

The polarized neutron imaging technique provides a non-invasive method of characterizing localized magnetic fields inside superconductors. However, complete understanding of the magnetic field distribution has yet to be realized experimentally due to the complexity of the interaction between neutron polarization and magnetic field. In this article, we show that a well-defined and controlled magnetic field through the neutron path contributes to simplify the data analysis and makes future quantitative polarized neutron imaging possible. This is demonstrated in a set of experiments that visualize the magnetic field distribution inside and around the superconductors. The experimental results demonstrate that proper guide field setup allows the visualization of the magnetic field expulsion at the surface of the superconductor in the zero-field cooling condition, as well as the magnetic field trapped inside the superconductor under field cooling condition.

Cold Storage Exacerbates Renal and Mitochondrial Dysfunction Following Transplantation.

Long-term renal function is compromised in patients receiving deceased donor kidneys which require cold storage exposure prior to transplantation. It is well established that extended cold storage induces renal damage and several labs, including our own, have demonstrated renal mitochondrial damage after cold storage alone. However, to our knowledge, few studies have assessed renal and mitochondrial function after transplantation of rat kidneys exposed to short-term (4 hr) cold storage compared to transplant without cold storage (autotransplantation). Our data reveal that cold storage plus transplantation exacerbated renal and mitochondrial dysfunction when compared to autotransplantation alone.

Renal Mitochondrial Lipid Peroxidation during Sepsis.

Sepsis can provoke kidney injury, which increases mortality. Human and animal studies have documented increased renal oxidative injury and mitochondrial damage during sepsis. However, few studies have attempted to dissect specific renal targets and/or types of oxidative injury using the cecal ligation and puncture (CLP) murine model of sepsis. The purpose of this short communication is to examine the extent of lipid peroxidation within renal mitochondria using CLP and blue native gel electrophoresis which separates intact mitochondrial respiratory complexes. Our results show that CLP induced increased 4-hydroxy-nonenal protein adduction (marker of lipid peroxidation) in renal homogenates and mitochondrial fractions. Blue native gel electrophoresis revealed that respiratory complex III was selectively targeted within mitochondrial fractions. This supports our prior report showing renal complex III inactivation following CLP. Future studies will identify specific renal proteins within complex III that are modified during sepsis to provide mechanistic insight on how mitochondrial respiration is inhibited during sepsis.

Targeting mitochondrial oxidants may facilitate recovery of renal function during infant sepsis.

Sepsis-induced acute kidney injury (SAKI) is a frequent complication of infant sepsis that approximately doubles the mortality rate. The poor prognosis of these patients is a result of care that is mainly supportive, nontargeted, and usually begun only after symptoms of the systemic inflammatory response syndrome are observed. Preclinical studies from relevant rodent models of SAKI suggest that mitochondria-targeted antioxidants may be a new mode of therapy that could promote recovery.

In situ polarized 3He system for the Magnetism Reflectometer at the Spallation Neutron Source.

We report on the in situ polarized (3)He neutron polarization analyzer developed for the time-of-flight Magnetism Reflectometer at the Spallation Neutron Source at Oak Ridge National Laboratory. Using the spin exchange optical pumping method, we achieved a (3)He polarization of 76% ± 1% and maintained it for the entire three-day duration of the test experiment. Based on transmission measurements with unpolarized neutrons, we show that the average analyzing efficiency of the (3)He system is 98% for the neutron wavelength band of 2-5 Å. Using a highly polarized incident neutron beam produced by a supermirror bender polarizer, we obtained a flipping ratio of >100 with a transmission of 25% for polarized neutrons, averaged over the wavelength band of 2-5 Å. After the cell was depolarized for transmission measurements, it was reproducibly polarized and this performance was maintained for three weeks. A high quality polarization analysis experiment was performed on a reference sample of Fe/Cr multilayer with strong spin-flip off-specular scattering. Using a combination of the position sensitive detector, time-of-flight method, and the excellent parameters of the (3)He cell, the polarization analysis of the two-dimensional maps of reflected, refracted, and off-specular scattered intensity above and below the horizon were obtained, simultaneously.

Nitration of manganese superoxide dismutase during ocular inflammation.

Reactive nitrogen species, in particular, peroxynitrite (ONOO(-)) have been proposed to play an important role in the pathogenesis of endotoxin-induced uveitis (EIU). Tyrosine nitration by ONOO(-) has been shown in other model systems to inhibit the activity of the superoxide anion quenching enyzme, manganese superoxide dismutase (MnSOD), perhaps contributing to progression of disease. In this study, it is confirmed through immunoanalysis that nitrated proteins are produced during EIU, and furthermore, that MnSOD is a target of nitration during the inflammatory response. In addition, through microsequencing analyses, nitrated albumin--apparent in both control and EIU eyes--was identified. Positive immunostaining of nitrated proteins was seen in the ciliary epithelium, inflammatory cells, and protein exudate of eyes from rats injected with endotoxin. Incubation of nitrotyrosine immunoprecipitates from the iris and ciliary body (ICB) with a polyclonal antibody against MnSOD revealed that nitrated MnSOD was present only in the ICB of EIU rats. When the total activity of the enzyme was examined, it was observed that despite the presence of nitrated MnSOD, activity was increased relative to control. Analysis of MnSOD mRNA and protein from the ICB of both groups demonstrated an increase in mRNA expression and consequently a three- to five-fold increase in MnSOD protein in EIU rats as compared to control rats. Further examination of MnSOD protein expression through immunohistochemistry noted enhanced immunostaining in the ciliary epithelium of eyes of EIU rats. Additional investigation of a 70 kDa band apparent in nitrotyrosine immunoprecipitates from the ICB of control and EIU rats revealed that the plasma protein albumin is nitrated as well. This protein is present as a result of the breakdown of the blood-aqueous barrier during inflammation. In summary, two endogenous nitration targets, albumin and MnSOD, were identified. Nitrated MnSOD appears to be specifically targeted to the ICB during inflammation, underscoring the importance of the interface in EIU. Furthermore, the expression and activity of the enzyme is increased in the ICB during EIU, perhaps regulating reactive nitrogen species produced within the cells. This study implicates ONOO(-) in the pathogenesis of EIU and imparts the putative role MnSOD plays in disease resolution.

Mitochondrial tyrosine nitration precedes chronic allograft nephropathy.

Endogenous tyrosine nitration and inactivation of manganese superoxide dismutase (MnSOD) has previously been reported to occur during end-stage human renal allograft rejection. In order to determine whether nitration and inactivation of this critical mitochondrial protein might play a contributory role in the onset of transplant rejection, we employed a rodent model of Chronic Allograft Nephropathy (or CAN). Using this model we followed kidney function from 2-52 weeks post-transplant and correlated graft function with levels of nitration in the renal allograft. Tyrosine nitration of both glomerular and tubular structures occurred at 2 weeks post-transplant. At later times (16 weeks) post-transplant, tyrosine nitration appeared to be confined to tubular structures; however glomerular nitration returned at 52 weeks post-transplant. Interestingly, nitration and inactivation of MnSOD occurs prior to the onset of renal dysfunction in this rat model of chronic allograft nephropathy (2 weeks versus 16 weeks post-transplant). Furthermore, we have identified an additional mitochondrial protein, cytochrome c, as being endogenously nitrated during chronic rejection. The kinetics of cytochrome c nitration lagged behind MnSOD nitration and inactivation (4 weeks compared to 2 weeks); suggesting that loss of MnSOD activity likely contributes to elevation of the nitrating species and further nitration of other targets.

Evidence for peroxynitrite-mediated modifications to p53 in human gliomas: possible functional consequences.

Based on previous findings of increased nitric oxide synthase (NOS) expression in human gliomas (4), we hypothesized that peroxynitrite, a highly reactive metabolite of nitric oxide (NO) and superoxide (O(*-)(2)), might be increased in these tumors in vivo. Here we demonstrate that nitrotyrosine (a footprint of peroxynitrite protein modification) is present in human malignant gliomas. Furthermore, we show that p53, a key tumor suppressor protein, has evidence of peroxynitrite-mediated modifications in gliomas in vivo. Experiments in vitro demonstrate that peroxynitrite treatment of recombinant wild-type p53 at physiological concentrations results in formation of higher molecular weight aggregates, tyrosine nitration, and loss of specific DNA binding. Peroxynitrite treatment of human glioma cell lysates similarly resulted in selective tyrosine nitration of p53 and was also associated with loss of p53 DNA binding ability. These data indicate that tyrosine nitration of proteins occurs in human gliomas in vivo, that p53 may be a target of peroxynitrite in these tumors, and that physiological concentrations of peroxynitrite can result in a loss of p53 DNA binding ability in vitro. These findings raise the possibility that peroxynitrite may contribute to loss of wild-type p53 functional activity in gliomas by posttranslational protein modifications.

Invited review: manganese superoxide dismutase in disease.

Manganese superoxide dismutase (MnSOD) is essential for life as dramatically illustrated by the neonatal lethality of mice that are deficient in MnSOD. In addition, mice expressing only 50% of the normal compliment of MnSOD demonstrate increased susceptibility to oxidative stress and severe mitochondrial dysfunction resulting from elevation of reactive oxygen species. Thus, it is important to know the status of both MnSOD protein levels and activity in order to assess its role as an important regulator of cell biology. Numerous studies have shown that MnSOD can be induced to protect against pro-oxidant insults resulting from cytokine treatment, ultraviolet light, irradiation, certain tumors, amyotrophic lateral sclerosis, and ischemia/reperfusion. In addition, overexpression of MnSOD has been shown to protect against pro-apoptotic stimuli as well as ischemic damage. Conversely, several studies have reported declines in MnSOD activity during diseases including cancer, aging, progeria, asthma, and transplant rejection. The precise biochemical/molecular mechanisms involved with this loss in activity are not well understood. Certainly, MnSOD gene expression or other defects could play a role in such inactivation. However, based on recent findings regarding the susceptibility of MnSOD to oxidative inactivation, it is equally likely that post-translational modification of MnSOD may account for the loss of activity. Our laboratory has recently demonstrated that MnSOD is tyrosine nitrated and inactivated during human kidney allograft rejection and human pancreatic ductal adenocarcinoma. We have determined that peroxynitrite (ONOO- ) is the only known biological oxidant competent to inactivate enzymatic activity, to nitrate critical tyrosine residues, and to induce dityrosine formation in MnSOD. Tyrosine nitration and inactivation of MnSOD would lead to increased levels of superoxide and concomitant increases in ONOO- within the mitochondria which, could lead to tyrosine nitration/oxidation of key mitochondrial proteins and ultimately mitochondrial dysfunction and cell death. This article assesses the important role of MnSOD activity in various pathological states in light of this potentially lethal positive feedback cycle involving oxidative inactivation.

Acidic fibroblast growth factor (FGF-1) signaling inhibits peroxynitrite-induced cell death during pancreatic tumorigenesis.

Previous immunohistochemical studies have demonstrated enhanced appearance of FGF-1 and nitrotyrosine, a footprint of reactive nitrogen species peroxynitrite (ONOO(-)), in human pancreatic adenocarcinoma. We have examined the consequences of constitutive exposure to FGF-1 in nontumorigenic rat ductal epithelial cells (ARIP). ARIP cells were transduced with either a secreted chimera of FGF-1, ARIP(FGF-1), or a control plasmid, 65 RIP(betag). These cells were evaluated for alteration in growth and morphology, responses to ONOO(-) (protein tyrosine nitration/phosphorylation), and in vivo tumor formation. ARIP(FGF-1) cells, in contrast to 65 RIP(betag), demonstrated a transformed morphology, a 2-fold increased growth rate, and enhanced protein tyrosine phosphorylation. Treatment with 150 microM ONOO(-) resulted in 86 and 7% (p <.01) death of ARIP(betag) and ARIP(FGF-1), respectively. Exposure of 65 RIP(betag) cells to ONOO(-) enhanced tyrosine phosphorylation and tyrosine nitration of several polypeptides. Cell signaling by FGF-1 enhanced both phosphorylation and nitration of tyrosine residues in target proteins modified by ONOO(-). ARIP(betag) cells failed to exhibit tumor formation in nude mice, but at d 7 in vivo cells were TUNEL and nitrotyrosine positive and FGF-1 negative. ARIP(FGF-1) cells readily formed tumor nodules, exhibiting features of pancreatic adenocarcinoma and demonstrating FGF-1-positive, nitrotyrosine-positive, and TUNEL-negative epithelium. These results suggest an interdependent role between FGF-1 and ONOO(-) during the development and progression of pancreatic adenocarcinoma.

Increased dietary salt accelerates chronic allograft nephropathy in rats.

BACKGROUND: Chronic allograft nephropathy (CAN), a major problem in renal transplantation, is related to both alloantigen-dependent and -independent processes. Because dietary salt intake modulated glomerular production of transforming growth factor-beta, which has been shown to play an important role in CAN, we hypothesized that dietary salt would directly enhance renal injury in a rodent model of CAN. METHODS: Dietary NaCl was increased from 1.0% (normal) to 8.0% in a group of Fisher/Lewis rats 25 days following orthotopic renal transplantation and was continued until 16 weeks after transplantation. RESULTS: Blood pressure, which was recorded using radiotelemetry in the first eight-weeks post-transplantation, did not differ between the groups, but allograft recipients on the 8.0% NaCl diet rapidly demonstrated increased urinary albumin excretion. Renal function determined by dynamic functional imaging was worse in allograft recipients on the 8.0% NaCl diet by six weeks following transplantation. Histologic examination at 16 weeks confirmed a significant increase in allograft damage in the 8.0% NaCl group compared with allografts from rats on 1.0% NaCl diet. These findings included glomerulosclerosis and tubulointerstitial injury that consisted of fibrosis, tubular atrophy and dilation, intratubular casts, and tubular epithelial cell damage. Small arteries and arterioles did not show evidence of damage from hypertension or other abnormality. CONCLUSIONS: In this model of CAN, renal allograft dysfunction preceded hypertension and was accelerated significantly by an increase in dietary salt.

Tyrosine nitration of c-SRC tyrosine kinase in human pancreatic ductal adenocarcinoma.

During pancreatic tumorigenesis, the equilibrium between cell survival and cell death is altered, allowing aggressive neoplasia and resistance to radiation and chemotherapy. Local oxidative stress is one mechanism regulating programmed cell death and growth and may contribute to both tumor progression and suppression. Our recent in situ immunohistochemical studies demonstrated that levels of total nitrotyrosine, a footprint of the reactive nitrogen species peroxynitrite, are elevated in human pancreatic ductal adenocarcinomas. In this study, quantitative HPLC-EC techniques demonstrated a 21- to 97-fold increase in the overall levels of nitrotyrosine of human pancreatic tumor extracts compared to normal pancreatic extracts. Western blot analysis of human pancreatic tumor extracts showed that tyrosine nitration was restricted to a few specific proteins. Immunoprecipitation coupled with Western analysis identified c-Src tyrosine kinase as a target of both tyrosine nitration and tyrosine phosphorylation. Peroxynitrite treatment of human pancreatic carcinoma cells in vitro resulted in increased tyrosine nitration and tyrosine phosphorylation of c-Src kinase, increased (>2-fold) c-Src kinase activity, and increased association between c-Src kinase and its downstream substrate cortactin. Collectively, these observations suggest that peroxynitrite-mediated tyrosine nitration and tyrosine phosphorylation of c-Src kinase may lead to enhanced tyrosine kinase signaling observed during pancreatic ductal adenocarcinoma growth and metastasis.

Rapid and irreversible inactivation of protein tyrosine phosphatases PTP1B, CD45, and LAR by peroxynitrite.

Protein tyrosine phosphatases (PTPs) contain an essential thiol in the active site which may be susceptible to attack by nitric oxide-derived biological oxidants. We assessed the effects of peroxynitrite, nitric oxide, and S-nitrosoglutathione on the activity of three human tyrosine phosphatases in vitro. The receptor-like T-cell tyrosine phosphatase (CD45), the non-receptor-like tyrosine phosphatase PTP1B, and leukocyte-antigen-related (LAR) phosphatase were all irreversibly inactivated by peroxynitrite in less than 1 s with IC(50) values of

Tyrosine modifications and inactivation of active site manganese superoxide dismutase mutant (Y34F) by peroxynitrite.

Recent studies from this laboratory have demonstrated that human manganese superoxide dismutase (MnSOD) is a target for tyrosine nitration in several chronic inflammatory diseases including chronic organ rejection, arthritis, and tumorigenesis. Furthermore, we demonstrated that peroxynitrite (ONOO-) is the only known biological oxidant competent to inactivate enzymatic activity, nitrate critical tyrosine residues, and induce dityrosine formation in MnSOD. To elucidate the differential contributions of tyrosine nitration and oxidation during enzymatic inactivation, we now compare ONOO- treatment of native recombinant human MnSOD (WT-MnSOD) and a mutant, Y34F-MnSOD, in which tyrosine 34 (the residue most susceptible to ONOO--mediated nitration) was mutated to phenylalanine. Both WT-MnSOD (IC50 = 65 microM, 15 microM MnSOD) and Y34F-MnSOD (IC50 = 55 microM, 15 microM Y34F) displayed similar dose-dependent sensitivity to ONOO--mediated inactivation. Compared to WT-MnSOD, the Y34F-MnSOD mutant demonstrated significantly less efficient tyrosine nitration and enhanced formation of dityrosine following treatment with ONOO-. Collectively, these results suggest that complete inactivation of MnSOD by ONOO- can occur independent of the active site tyrosine residue and includes not only nitration of critical tyrosine residues but also tyrosine oxidation and subsequent formation of dityrosine.

Association of increased immunostaining for inducible nitric oxide synthase and nitrotyrosine with fibroblast growth factor transformation in pancreatic cancer.

BACKGROUND: Despite recognition of the devastating malignant potential of pancreatic cancer, the exact pathophysiological events contributing to tumor growth, vascular invasiveness, and hepatic metastasis remain to be elucidated. METHODS: Twelve human pancreatic adenocarcinomas were evaluated using immunohistochemical and in situ hybridization techniques for the appearance of the angiogenic and neurogenic growth factors, acidic fibroblast (FGF-1) and basic fibroblast growth factor (FGF-2), and their high-affinity receptors. Since FGF biological processes appear to be regulated by oxidant stress, tumors were examined further for the immunoappearance of inducible nitric oxide synthase (iNOS) and nitrotyrosine. RESULTS: Compared with normal human pancreatic tissue, tumor specimens exhibited varying levels of enhanced staining for FGF ligands and receptors. The increased appearance of FGF-1 and FGF-2 proteins was accompanied by increased detection of messenger RNA encoding each growth factor. In addition, these pancreatic tumors demonstrated the overexpression of iNOS and immunostaining of nitrotyrosine compared with normal pancreatic tissue. CONCLUSIONS: The enhanced expression of FGF and FGF receptors suggests that these polypeptide mitogens may serve as important mediators of growth and of angiogenic and metastatic responses associated with pancreatic tumors, not seen in normal pancreatic tissue. Furthermore, we provide the first indication of increased expression of iNOS and protein tyrosine nitration, thereby predicting the potential involvement of oxidant stress during development and progression of pancreatic adenocarcinoma.

Relative hand skill predicts academic ability: global deficits at the point of hemispheric indecision.

Population variation in handedness (a correlate of cerebral dominance for language) is in part genetic and, it has been suggested, its persistence represents a balanced polymorphism with respect to cognitive ability. This hypothesis was tested in a sample of 12,770 individuals in a UK national cohort (the National Child Development Study) by assessing relative hand skill (in a square checking task) as a predictor of verbal, non-verbal, and mathematical ability and reading comprehension at the age of 11 years. Whereas some modest decrements were present in extreme right handers the most substantial deficits in ability were seen close to the point of equal hand skill ('hemispheric indecision'). For verbal ability females performed better than males, but the relationship to relative hand skill was closely similar for the two sexes; for reading comprehension males close to the point of equal hand skill showed greater impairments than females. Analysed by writing hand the relationship of ability to hand skill appeared symmetrical about the point of 'hemispheric indecision'. The variation associated with degrees of dominance may reflect the operation of continuing selection on the gene (postulated to be X-Y linked) by which language evolved and speciation occurred.

Peroxynitrite-mediated inactivation of manganese superoxide dismutase involves nitration and oxidation of critical tyrosine residues.

Previous studies from our laboratory have demonstrated that the mitochondrial protein manganese superoxide dismutase is inactivated, tyrosine nitrated, and present as higher molecular mass species during human renal allograft rejection. To elucidate mechanisms whereby tyrosine modifications might result in loss of enzymatic activity and altered structure, the effects of specific biological oxidants on recombinant human manganese superoxide dismutase in vitro have been evaluated. Hydrogen peroxide or nitric oxide had no effect on enzymatic activity, tyrosine modification, or electrophoretic mobility. Exposure to either hypochlorous acid or tetranitromethane (pH 6) inhibited (approximately 50%) enzymatic activity and induced the formation of dityrosine and higher mass species. Treatment with tetranitromethane (pH 8) inhibited enzymatic activity 67% and induced the formation of nitrotyrosine. In contrast, peroxynitrite completely inhibited enzymatic activity and induced formation of both nitrotyrosine and dityrosine along with higher molecular mass species. Combination of real-time spectral analysis and electrospray mass spectroscopy revealed that only three (Y34, Y45, and Y193) of the nine total tyrosine residues in manganese superoxide dismutase were nitrated by peroxynitrite. Inspection of X-ray crystallographic data suggested that neighboring glutamate residues associated with two of these tyrosines may promote targeted nitration by peroxynitrite. Tyr34, which is present in the active site, appeared to be the most susceptible residue to peroxynitrite-mediated nitration. Collectively, these observations are consistent with previous results using chronically rejecting human renal allografts and provide a compelling argument supporting the involvement of peroxynitrite during this pathophysiologic condition.