Background Pembrolizumab demonstrated robust antitumor activity and safety in the phase Ib KEYNOTE-001 study (NCT01295827) of advanced melanoma. Five-year outcomes in all patients and treatment-naive patients are reported herein. Patients whose disease progressed following initial response and who received a second course of pembrolizumab were also analyzed. Patients and methods Patients aged ≥18 years with previously treated or treatment-naive advanced/metastatic melanoma received pembrolizumab 2 mg/kg every 3 weeks, 10 mg/kg every 3 weeks, or 10 mg/kg every 2 weeks until disease progression, intolerable toxicity, or patient/investigator decision to withdraw. Kaplan–Meier estimates of overall survival (OS) and progression-free survival (PFS) were calculated. Objective response rate and PFS were based on immune-related response criteria by investigator assessment (data cut-off, September 1, 2017). Results KEYNOTE-001 enrolled 655 patients with melanoma; median follow-up was 55 months. Estimated 5-year OS was 34% in all patients and 41% in treatment-naive patients; median OS was 23.8 months (95% CI, 20.2–30.4) and 38.6 months (95% CI, 27.2–not reached), respectively. Estimated 5-year PFS rates were 21% in all patients and 29% in treatment-naive patients; median PFS was 8.3 months (95% CI, 5.8–11.1) and 16.9 months (95% CI, 9.3–35.5), respectively. Median response duration was not reached; 73% of all responses and 82% of treatment-naive responses were ongoing at data cut-off; the longest response was ongoing at 66 months. Four patients [all with prior response of complete response (CR)] whose disease progressed during observation subsequently received second-course pembrolizumab. One patient each achieved CR and partial response (after data cut-off). Treatment-related AEs (TRAEs) occurred in 86% of patients and resulted in study discontinuation in 7.8%; 17% experienced grade 3/4 TRAE. Conclusions This 5-year analysis of KEYNOTE-001 represents the longest follow-up for pembrolizumab to date and confirms the durable antitumor activity and tolerability of pembrolizumab in advanced melanoma. Clinical Trial Registry ClinicalTrials.gov, NCT01295827.

Recent studies suggest that manganese-induced neurodegenerative toxicity may be partly due to its action on aconitase, which participates in cellular iron regulation and mitochondrial energy production. This study was performed to investigate whether chronic manganese exposure in rats influenced the homeostasis of iron in blood and cerebrospinal fluid (CSF). Groups of 8-10 rats received intraperitoneal injections of MnCl2 at the dose of 6 mg Mn/kg/day or equal volume of saline for 30 days. Concentrations of manganese and iron in plasma and CSF were determined by atomic absorption spectrophotometry. Rats exposed to manganese showed a greatly elevated manganese concentration in both plasma and CSF. The magnitude of increase in CSF manganese (11-fold) was equivalent to that of plasma (10-fold). Chronic manganese exposure resulted in a 32% decrease in plasma iron (p<0.01) and no changes in plasma total iron binding capacity (TIBC). However, it increased CSF iron by 3-fold as compared to the controls (p<0.01). Northern blot analyses of whole brain homogenates revealed a 34% increase in the expression of glutamine synthetase (p<0.05) with unchanged metallothionein-I in manganese-intoxicated rats. When the cultured choroidal epithelial cells derived from rat choroid plexus were incubated with MnCl2 (100 microM) for four days, the expression of transferrin receptor mRNA appeared to exceed by 50% that of control (p<0.002). The results indicate that chronic manganese exposure alters iron homeostasis possibly by expediting unidirectional influx of iron from the systemic circulation to cerebral compartment. The action appears likely to be mediated by manganese-facilitated iron transport at brain barrier systems.

The choroid plexus plays a wide range of roles in brain development, maturation, aging process, endocrine regulation, and pathogenesis of certain neurodegenerative diseases. To facilitate in vitro study, we have used a gene transfection technique to immortalize murine choroidal epithelial cells. A viral plasmid (pSV3neo) was inserted into the host genome of primary choroidal epithelia by calcium phosphate precipitation. The transfected epithelial cells, i.e., Z310 cells, that survived from cytotoxic selection expressed SV40 large-T antigen throughout the life span, suggesting a successful gene transfection. The cells displayed the same polygonal epithelial morphology as the starting cells by light microscopy. Immunocytochemical studies demonstrate the presence of transthyretin (TTR), a thyroxine transport protein known to be exclusively produced by the choroidal epithelia in the CNS, in both transfected and starting cells. Western blot analyses further confirm the production and secretion of TTR by these cells. The mRNAs encoding transferrin receptor (TfR) were identified by Northern blot analyses. The cells grow at a steady rate, currently in the 110th passage with a population doubling time of 20-22 h in the established culture. When Z310 cells were cultured onto a Trans-well apparatus, the cells formed an epithelial monolayer similar to primary choroidal cells, possessing features such as an uneven fluid level between inner and outer chambers and an electrical resistance approximately 150-200 omega-cm(2). These results indicate that immortalized Z310 cells possess the characteristics of choroidal epithelia and may have the potential for application in blood-CSF barrier (BCB) research.

Manganese (Mn)-induced neurodegenerative toxicity has been associated with a distorted iron (Fe) metabolism at both systemic and cellular levels. In the current study, we examined whether the oxidation states of Mn produced differential effects on certain mitochondrial [Fe-S] containing enzymes in vitro. When mitochondrial aconitase, which possesses a [4Fe-4S] cluster, was incubated with either Mn(II) or Mn(III), both Mn species inhibited the activities of aconitase. However, the IC 10 (concentration to cause a 10% enzyme inhibition) for Mn(III) was ninefold lower than that for Mn(II). Following exposure of mitochondrial fractions with Mn(II) or Mn(III), there was a significant inhibition by either Mn species in activities of Complex I whose active site contains five to eight [Fe-S] clusters. The dose-time response curves reveal that Mn(III) was more effective in blocking Complex I activity than Mn(II). Northern blotting was used to examine the expression of mRNAs encoding transferrin receptor (TfR), which is regulated by cytosolic aconitase. Treatment of cultured PC12 cells with Mn(II) and Mn(III) at 100 μM for 3 days resulted in 21 and 58% increases, respectively, in the expression of TfR mRNA. Further studies on cell growth dynamics after exposure to 25-50 μM Mn in culture media demonstrated that the cell numbers were much reduced in Mn(III)-treated groups compared to Mn(II)-treated groups, suggesting that Mn(III) is more effective than Mn(II) in cell killing. In cells exposed to Mn(II) and Mn(III), mitochondrial DNA (mtDNA) was significantly decreased by 24 and 16%, respectively. In contrast, rotenone and MPP+ did not seem to alter mtDNA levels. These in vitro results suggest that Mn(III) species appears to be more cytotoxic than Mn(II) species, possibly due to higher oxidative reactivity and closer radius resemblance to Fe. Keywords manganese; iron; aconitase; Complex I; speciation; transferrin receptor; PC12 cells; mitochondria; mitochondrial DNA; cytotoxicity; Fe-S cluster Chronic manganese (Mn) intoxication in humans causes permanent neurodegenerative damage in the nigrostriatal region, resulting in a syndrome similar to Parkinson's disease (PD;Cook et al., 1974;Mena et al., 1967 Jenner et al., 1992;Sofic et al., 1991;Ye et al., 1996). A recent population study has also established that serum Fe concentrations are significantly reduced in IPD patients compared with controls, suggesting a compartment shift in Fe from blood to tissues, including brain (Logroscino et al., 1997). The role of Fe in etiopathology of IPD has been extensively reviewed by Jenner et al. (1992) and Youdim et al. (1993).Previous studies from this laboratory have established that Mn-induced neurotoxicities appear to be associated with its interaction with Fe at systemic and cellular levels (Zheng et al., 1998Zheng and Zhao, 2001). Following Mn exposure, there is a predominant influx of Fe from the blood into the cerebrospinal fluid (CSF) and from extracellular matrix to intracellular space Zheng and Zhao, 2001 et al., 1983...

Our previous studies show that manganese (Mn) exposure inhibits aconitase, an enzyme regulating the proteins responsible for cellular iron (Fe) equilibrium. This study was performed to investigate whether Mn intoxication leads to an altered cellular Fe homeostasis in cultured neuronal or neuroglial cells as a result of disrupted Fe regulation. Our results reveal a significant increase in the expression of transferrin receptor (TfR) mRNAs and a corresponding increase in cellular 59 Fe net uptake by PC12 cells, but not astrocytes, following Mn exposure. These findings suggest that alteration by Mn of cellular Fe homeostasis may contribute to Mn-induced neuronal cytotoxicity. KeywordsManganese; Iron; Transferrin receptor; Transferrin receptor mRNA; uptake; PC12 cell; Astrocyte Intracellular iron homeostasis is post-translationally regulated by one of the iron regulatory proteins (IRPs), namely cytoplasmic aconitase (ACO1) or IRP-I. This protein contains a unique [4Fe-4S] cubane cluster in its active catalytic site, with one particularly labile Fe atom. ACO1 can selectively bind to mRNAs containing a stem-loop structure, also referred to as iron responsive elements (IRE) [4,11]. In iron-replete cells, ACO1 secures iron as part of its structure in the form of a [4Fe-4S] cluster. While this form of ACO1 binds poorly to mRNA, it can enzymatically catalyze the conversion of bound citrate to isocitrate. When cellular iron levels are insufficient, ACO1 assumes a [3Fe-4S] configuration, loses its cluster and enzymatic activity, and is transformed into an mRNA-binding protein. In the latter state, the enzyme binds with high affinity to IRE-containing mRNAs, inhibits translation of those mRNAs whose IRE's are 5′ (e.g., ferritin, succinic dehydrogenase, mitochondrial aconitase), and stimulates the expression of those whose IRE's are 3′ (e.g., transferrin receptor). The net result of this RNA-protein interaction is an increase in cellular Fe uptake and a decrease in Fe storage [4,9,11].Our previous studies indicate that Mn exposure significantly alters cellular aconitase activity [19]. This may be primarily due to a close mimicry between Mn and Fe in their coordination chemistry, allowing Mn to compete with Fe and insert itself into the fourth, labile Fe binding site in the enzyme's active center. We postulate that such replacement, while suppressing ACO1's enzymatic catalytic function, would increase the protein's ability to bind to mRNAs encoding transferrin receptor (TfR), which in concert with a down-regulation of Fe storage . The cells were plated in 24-well (4 cm 2 /well) trays and incubated in RPMI 1640 medium (ATCC) with 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, 4.5 g/l glucose, 10 mM HEPES, 1.0 mM sodium pyruvate, 10% heat-inactivated horse serum, and 5% FBS. The medium was changed every 2-3 days. A primary culture of astrocytes was established according to the procedure described by Goodman et al., [7]. The cerebral cortices of newborn SpragueDawley rats (Hilltop, Cottdale, PA) were removed and minced wi...