Ibrutinib in combination with nab-paclitaxel and gemcitabine for first-line treatment of patients with metastatic pancreatic adenocarcinoma: phase III RESOLVE study.

BACKGROUND: First-line treatment of metastatic pancreatic ductal adenocarcinoma (PDAC) includes nab-paclitaxel/gemcitabine. Ibrutinib, a Bruton's tyrosine kinase inhibitor, exhibits antitumor activity through tumor microenvironment modulation. The safety and efficacy of first-line ibrutinib plus nab-paclitaxel/gemcitabine treatment in patients with PDAC were evaluated. PATIENTS AND METHODS: RESOLVE (NCT02436668) was a phase III, randomized, double-blind, placebo-controlled study. Patients (histologically-confirmed PDAC; stage IV diagnosis ≥6 weeks of randomization; Karnofsky performance score ≥70) were randomized to once-daily oral ibrutinib (560 mg) or placebo plus nab-paclitaxel (125 mg/m2) and gemcitabine (1000 mg/m2). Primary endpoints were overall survival (OS) and investigator-assessed progression-free survival (PFS); overall response rate and safety were assessed. RESULTS: In total, 424 patients were randomized (ibrutinib arm, n = 211; placebo arm, n = 213). Baseline characteristics were balanced across arms. After a median follow-up of 25 months, there was no significant difference in OS between ibrutinib plus nab-paclitaxel/gemcitabine versus placebo plus nab-paclitaxel/gemcitabine (median of 9.7 versus 10.8 months; P = 0.3225). PFS was shorter for ibrutinib plus nab-paclitaxel/gemcitabine compared with placebo plus nab-paclitaxel/gemcitabine (median 5.3 versus 6.0 months; P < 0.0001). Overall response rates were 29% and 42%, respectively (P = 0.0058). Patients in the ibrutinib arm had less time on treatment and received lower cumulative doses for all agents compared with the placebo arm. The most common grade ≥3 adverse events for ibrutinib versus placebo arms included neutropenia (24% versus 35%), peripheral sensory neuropathy (17% versus 8%), and anemia (16% versus 17%). Primary reasons for any treatment discontinuation were disease progression and adverse events. CONCLUSIONS: Ibrutinib plus nab-paclitaxel/gemcitabine did not improve OS or PFS for patients with PDAC. Safety was consistent with known profiles for these agents.

Prediction of Outcome in Patients With Chronic Lymphocytic Leukemia Treated With Ibrutinib: Development and Validation of a Four-Factor Prognostic Model.

PURPOSE: Randomized trials established the superiority of ibrutinib-based therapy over chemoimmunotherapy in chronic lymphocytic leukemia. Durability of progression-free survival (PFS) with ibrutinib can vary by patient subgroup. Clinical tools for prognostication and risk-stratification are needed. PATIENTS AND METHODS: Patients treated with ibrutinib in phase II and III trials provided the discovery data set and were subdivided into discovery and internal validation cohorts. An external validation cohort included 84 patients enrolled in our investigator-initiated phase II trial. Univariable analysis of 18 pretreatment parameters was performed using PFS and overall survival (OS) end-points. Multivariable analysis and machine-learning algorithms identified four factors for a prognostic model that was validated in internal and external cohorts. RESULTS: Factors independently associated with inferior PFS and OS were as follows: TP53 aberration, prior treatment, ß-2 microglobulin ≥ 5 mg/L, and lactate dehydrogenase > 250 U/L. Each of these four factors contributed one point to a prognostic model that stratified patients into three risk groups: three to four points, high risk; two points, intermediate risk; zero to one point, low risk. The 3-year PFS rates for all 804 patients combined were 47%, 74%, and 87% for the high-, the intermediate-, and the low-risk group, respectively (P < .0001). The 3-year OS rates were 63%, 83%, and 93%, respectively (P < .0001). The model remained significant when applied to treatment-naïve and relapsed/refractory cohorts individually. For 84 patients in the external cohort, BTK and PLCG2 mutations were tested cross-sectionally and at progression. The cumulative incidences of mutations were strongly correlated with the model. In the external cohort, Richter's transformation occurred in 17% of the high-risk group, and in no patient in the low-risk group. CONCLUSION: Patients at increased risk of ibrutinib failure can be identified at treatment initiation and considered for clinical trials.

Phase I Study of Enavatuzumab, a First-in-Class Humanized Monoclonal Antibody Targeting the TWEAK Receptor, in Patients with Advanced Solid Tumors.

This phase I study evaluates the safety, MTD, pharmacokinetics (PK), pharmacodynamics, and preliminary anticancer activity of enavatuzumab, a humanized IgG1 antibody to the TWEAK receptor, in patients with advanced solid malignancies. Patients received escalating doses of enavatuzumab given intravenously over 60 minutes every 2 weeks. Blood was obtained for PK and biomarker assessment. Three patients were enrolled per dose level in a standard 3+3 design with response assessment by RECIST version 1.0, every 8 weeks. Thirty patients were enrolled at 6 dose levels ranging from 0.1 to 1.5 mg/kg. Dose-limiting toxicities included grade 4 (G4) lipase, G3 bilirubin, and G4 amylase elevations. There was no apparent correlation of liver or pancreatic enzyme elevation with drug exposure or the presence of liver metastases. Enavatuzumab exhibited a two-compartment linear PK model. Estimated systemic clearance was 23 to 33 mL/h with an elimination half-life of 7 to 18 days. The predicted target efficacious peak and trough concentrations occurred at 1.0 mg/kg following the second dose. There were no objective responses; 4 patients had stable disease. The MTD of enavatuzumab is 1.0 mg/kg i.v. every 2 weeks. Higher doses were not tolerated due to hepatopancreatic lab abnormalities. Further evaluation of the mechanisms of the liver and pancreatic enzyme toxicities is needed before embarking on further single-agent or combination strategies. Mol Cancer Ther; 17(1); 215-21. ©2017 AACR.

Consistent efficacy of daclizumab beta across patient demographic and disease activity subgroups in patients with relapsing-remitting multiple sclerosis.

BACKGROUND: Daclizumab beta is a humanized monoclonal antibody specific for the human interleukin-2 receptor alpha chain (CD25). In two pivotal studies in relapsing multiple sclerosis (MS), patients treated with daclizumab beta exhibited lower annualized relapse rates (ARR) when compared with placebo or with intramuscular (IM) interferon beta-1a. OBJECTIVES: To determine if the efficacy of daclizumab beta demonstrated in the phase 2 SELECT study and the phase 3 DECIDE study was consistent in patient subgroups. METHODS: In the SELECT study, patients received daclizumab beta 150 or 300mg administered subcutaneously every 4 weeks for 52 weeks, and were compared with patients who received placebo. In the DECIDE study, patients received daclizumab beta 150mg administered subcutaneously every 4 weeks for 96-144 weeks, and were compared with patients who received IM interferon beta-1a 30µg. Subgroups were defined by sex, age, the number of relapses in the year before the study, disease duration, baseline disability measured by EDSS, presence of Gd-enhancing lesions, T2 hyperintense lesion volume at baseline, and previous interferon beta-1a use. RESULTS: Treatment with daclizumab beta was associated with relative lower ARR, with 95% confidence intervals (CIs) below 1 in 13 of 15 subgroups (SELECT study) compared with placebo and in all 17 subgroups compared with interferon beta-1a (DECIDE study). In 2 subgroups in the SELECT study (patients who were older than 35 years of age or who had a disease duration of 10 or more years), the rate ratio point estimate for the ARR was in favor of daclizumab beta but the 95% CI overlapped with 1. The clinical benefits in ARR achieved with daclizumab beta treatment compared with placebo or interferon beta-1a across subgroups were similarly supported by reductions in lesion activity on magnetic resonance images (MRIs). CONCLUSIONS: These findings suggest that treatment with daclizumab beta is consistently effective among clinically important patient subgroups and support its potential as a viable therapeutic option across the spectrum of relapsing MS.

Randomized phase 2 study: elotuzumab plus bortezomib/dexamethasone vs bortezomib/dexamethasone for relapsed/refractory MM.

In this proof-of-concept, open-label, phase 2 study, patients with relapsed/refractory multiple myeloma (RRMM) received elotuzumab with bortezomib and dexamethasone (EBd) or bortezomib and dexamethasone (Bd) until disease progression/unacceptable toxicity. Primary endpoint was progression-free survival (PFS); secondary/exploratory endpoints included overall response rate (ORR) and overall survival (OS). Two-sided 0.30 significance level was specified (80% power, 103 events) to detect hazard ratio (HR) of 0.69. Efficacy and safety analyses were performed on all randomized patients and all treated patients, respectively. Of 152 randomized patients (77 EBd, 75 Bd), 150 were treated (75 EBd, 75 Bd). PFS was greater with EBd vs Bd (HR, 0.72; 70% confidence interval [CI], 0.59-0.88; stratified log-rank P = .09); median PFS was longer with EBd (9.7 months) vs Bd (6.9 months). In an updated analysis, EBd-treated patients homozygous for the high-affinity FcγRIIIa allele had median PFS of 22.3 months vs 9.8 months in EBd-treated patients homozygous for the low-affinity allele. ORR was 66% (EBd) vs 63% (Bd). Very good partial response or better occurred in 36% of patients (EBd) vs 27% (Bd). Early OS results, based on 40 deaths, revealed an HR of 0.61 (70% CI, 0.43-0.85). To date, 60 deaths have occurred (28 EBd, 32 Bd). No additional clinically significant adverse events occurred with EBd vs Bd. Grade 1/2 infusion reaction rate was low (5% EBd) and mitigated with premedication. In patients with RRMM, elotuzumab, an immunostimulatory antibody, appears to provide clinical benefit without added clinically significant toxicity when combined with Bd vs Bd alone. Registered to ClinicalTrials.gov as NCT01478048.

Elotuzumab in combination with lenalidomide and dexamethasone in patients with relapsed multiple myeloma: final phase 2 results from the randomised, open-label, phase 1b-2 dose-escalation study.

BACKGROUND: Elotuzumab, an immunostimulatory monoclonal antibody targeting signalling lymphocytic activation molecule (SLAM) family member 7 (SLAMF7), selectively kills SLAMF7-expressing myeloma cells through direct activation and engagement of the innate immune system, and thus might have clinical benefit in the treatment of myeloma. In phase 1 of this phase 1b-2 study, 82% of patients with relapsed multiple myeloma who were given elotuzumab plus lenalidomide and dexamethasone achieved an overall response. Here we report the final phase 2 results. METHODS: We did this randomised, multicentre, open-label, dose-escalation study (1703) at 17 hospitals in the USA, Canada, France, and Germany. Patients aged at least 18 years with confirmed, relapsed multiple myeloma, Eastern Cooperative Oncology Group performance status 0-2, and one to three previous therapies but no previous lenalidomide were eligible for phase 2. We randomly assigned patients (1:1) to either 10 mg/kg or 20 mg/kg intravenous elotuzumab plus oral lenalidomide (25 mg) and dexamethasone (40 mg). We stratified patients on the basis of the number of previous therapies (one versus two or three), and status of previous treatment with immunomodulatory drugs (yes or no), and used permuted block randomisation with a block size of four. Treatment was given in 28-day cycles until disease progression or unacceptable toxic effects occurred (elotuzumab was given on days 1, 8, 15, and 22 for cycles 1 to 2 and days 1 and 15 for subsequent cycles; lenalidomide was given on days 1-21 and dexamethasone once per week). The primary endpoint was the proportion of patients who achieved an objective response according to International Myeloma Working Group criteria. Primary analyses were done in the intention-to-treat population, and safety was analysed in all patients who received at least one dose of study drugs. This study is registered with ClinicalTrials.gov, number NCT00742560. FINDINGS: Between Jan 4, 2010, and Dec 21, 2010, we recruited and randomly assigned 73 patients to elotuzumab (36 to 10 mg/kg, 37 to 20 mg/kg). At data cutoff (Jan 16, 2014), 13 patients remained on treatment (six on 10 mg/kg, seven on 20 mg/kg). 61 (84%) patients achieved an objective response (33 [92%] with 10 mg/kg, 28 [76%] with 20 mg/kg); 31 (42%) a very good partial response (17 [47%] with 10 mg/kg, 14 [38%] with 20 mg/kg); and 20 (27%) a partial response (10 [28%] with 10 mg/kg, 10 [27%] with 20 mg/kg). The most common treatment-emergent adverse events of any grade were diarrhoea (48 [66%]), muscle spasms (45 [62%]), and fatigue (41 [56%]). 57 (78%) patients had grade 3-4 events, the most common of which were lymphopenia (15 [21%]) and neutropenia (14 [19%]). Three deaths occurred, none related to the study drugs. INTERPRETATION: Elotuzumab combined with lenalidomide and dexamethasone in patients with relapsed multiple myeloma showed acceptable safety and efficacy that seems better than that previously noted with lenalidomide and dexamethasone only. Phase 3 trials are in progress. FUNDING: Bristol-Myers Squibb, AbbVie Biotherapeutics.

BCAS2 promotes prostate cancer cells proliferation by enhancing AR mRNA transcription and protein stability.

BACKGROUND: We showed previously that breast carcinoma amplified sequence 2 (BCAS2) functions as a negative regulator of p53. We also found that BCAS2 is a potential AR-associated protein. AR is essential for the growth and survival of prostate carcinoma. Therefore we characterised the correlation between BCAS2 and AR. METHODS: Protein interactions were examined by GST pull-down assay and co-immunoprecipitation. Clinical prostate cancer (PCa) specimens were evaluated by immunohistochemical assay. AR transcriptional activity and LNCaP cell growth were assessed by luciferase assay and MTT assay, respectively. RESULTS: BCAS2 expression was significantly increased in PCa. BCAS2 stabilised AR protein through both hormone-dependent and -independent manners. There are at least two mechanisms for BCAS2-mediated AR protein upregulation: One is p53-dependent. The p53 is suppressed by BCAS2 that results in increasing AR mRNA and protein expression. The other is via p53-independent inhibition of proteasome degradation. As BCAS2 can form a complex with AR and HSP90, it may function with HSP90 to stabilise AR protein from being degraded by proteasome. CONCLUSIONS: In this study, we show that BCAS2 is a novel AR-interacting protein and characterise the correlation between BCAS2 and PCa. Thus we propose that BCAS2 could be a diagnostic marker and therapeutic target for PCa.

Effect of Sn addition on the corrosion behavior of Ti-7Cu-Sn cast alloys for biomedical applications.

The aim of this study was to investigate the effects of Sn content on the microstructure and corrosion resistance of Ti7CuXSn (x=0-5 wt.%) samples. The corrosion tests were carried out in 0.9 wt.% NaCl solution at 25 °C. The electrochemical corrosion behavior of the Ti7CuXSn alloy samples was evaluated using potentiodynamic polarization curves, electrochemical impedance spectroscopy (EIS), and equivalent circuit analysis. The resulting impedance parameters and polarization curves showed that adding Sn improved the electrochemical corrosion behavior of the Ti7CuXSn alloy. The Ti7CuXSn alloy samples were composed of a dual-layer oxide consisting of an inner barrier layer and an outer porous layer.

Phase Ib safety and pharmacokinetic study of volociximab, an anti-α5ß1 integrin antibody, in combination with carboplatin and paclitaxel in advanced non-small-cell lung cancer.

BACKGROUND: This phase Ib study evaluated volociximab, an anti-α5ß1 integrin antibody, in combination with carboplatin (Eli Lilly and Co., Indianapolis, IN) and paclitaxel (Taxol) in advanced, untreated non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: Three cohorts were treated with volociximab (10, 20, or 30 mg/kg) for up to six 3-week cycles in combination with carboplatin-paclitaxel chemotherapy and continued as maintenance therapy for patients with stable disease (SD) or better. Dose-limiting toxic effects, adverse events (AEs), pharmacokinetics, and anti-volociximab antibodies were assessed. RESULTS: A maximum tolerated dose was not reached up to the maximum planned dose of 30 mg/kg. In 29 patients who received volociximab, the most common grade≥3 AEs were neutropenia (24%), hyponatremia (17%), and fatigue (10%). Three patients experienced volociximab-related serious AEs. No hemorrhages were observed. Of 33 patients enrolled, 8 (24%) achieved a partial response and 17 (52%) had SD. The median progression-free survival was 6.3 months (95% confidence interval 5.5-8.1). Levels of potential biomarkers of angiogenesis or metastasis were reduced following six cycles of treatment. CONCLUSIONS: Volociximab combined with carboplatin and paclitaxel was generally well-tolerated and showed preliminary evidence of efficacy in advanced NSCLC.

Elotuzumab in combination with lenalidomide and low-dose dexamethasone in relapsed or refractory multiple myeloma.

PURPOSE: This phase I study evaluated elotuzumab, lenalidomide, and dexamethasone in patients with relapsed or refractory multiple myeloma (MM). PATIENTS AND METHODS: Three cohorts were enrolled and treated with elotuzumab (5.0, 10, or 20 mg/kg intravenously) on days 1, 8, 15, and 22 of a 28-day cycle in the first two cycles, and days 1 and 15 of each subsequent cycle; lenalidomide 25 mg orally [PO] on days 1 to 21; and dexamethasone 40 mg PO weekly. Dose-limiting toxicities (DLTs) were assessed during cycle 1 of each cohort, and clinical responses were evaluated during each cycle. The first five patients received up to six cycles of therapy; subsequent patients were treated until disease progression. RESULTS: Twenty-nine patients with advanced MM and a median of three prior MM therapies were enrolled; 28 patients were treated, three each in the 5.0-mg/kg and 10-mg/kg cohorts and 22 in the 20-mg/kg cohort. No DLTs were observed up to the maximum proposed dose of 20 mg/kg. The most frequent grade 3 to 4 toxicities were neutropenia (36%) and thrombocytopenia (21%). Two patients experienced a serious infusion reaction (one grade 4 anaphylactic reaction and one grade 3 stridor) during the first treatment cycle. Objective responses were obtained in 82% (23 of 28) of treated patients. After a median of 16.4 months follow-up, the median time to progression was not reached for patients in the 20-mg/kg cohort who were treated until disease progression. CONCLUSION: The combination of elotuzumab, lenalidomide, and low-dose dexamethasone was generally well tolerated and showed encouraging response rates in patients with relapsed or refractory MM.

[A synthesis of cinnamoyloxyisoflavones].

Interaction of 7-hydroxyisoflavonones with cinnamoyl chloride results in cinnamoyloxyisoflavonones.

Suicide: a literature review and its implications for nursing practice in Taiwan.

In recent years the suicide rates have been increasing gradually in many countries. In order to reduce the number of suicides, further research on suicide and the nursing care of suicidal people is required to enhance and advance the quality of suicide nursing care provided. Statistical evidence shows that the most common method of completing suicide in many countries is hanging. Other evidence demonstrates that some suicides could be prevented if all patients were assessed for suicide risk and if psychiatric nurses provided effective nursing care, which centres on therapeutic communication skills. This paper explores the literature on suicide and on the nursing care of people who are suicidal, and also on the importance of integrating theory with practice.

Nursing people who are suicidal on psychiatric wards in Taiwan: action/interaction strategies.

Suicide is a major mental health problem in Taiwan. Estimations revealed that approximately 41% of people who committed suicide had a previous history of psychiatric inpatient care. To date, a suicide nursing care theory has not been developed. Consequently, the aim of this study was to formulate a suicide nursing care theory with the aim of enhancing and advancing the nursing care provided to people who attempt suicide or have suicidal thoughts. A qualitative approach using grounded theory was adopted. A total of 15 peoples who had either suicidal ideas or had attempted suicide and 15 psychiatric nurses were interviewed and observed. Data were analysed using open, axial and selective coding and the NUD*IST software program. A substantive theory of suicide nursing care was developed from the emergent findings. Four categories surfaced in the nursing care theory relating to the nurses' 'action/interaction strategies'. They were: the holistic assessment of people who are suicidal; providing protection; providing basic care; and providing advanced care. The findings from this study could be used to influence and advance nurse education and training, clinical practice, management and further research.

In vitro anti-inflammatory effects of quercetin 3-O-methyl ether and other constituents from Rhamnus species.

The anti-inflammatory activities of the isolated flavonoids, quercetin 3-O-methyl ether (1), kaempferol (2), and quercetin (3), of Rhamnus nakaharai, and anthraquinone, frangulin B (4), of Rhamnus formosana, were assessed in vitro by determining their inhibitory effects on the chemical mediators released from mast cells, neutrophils, macrophages, and microglial cells. Compounds 1 - 3 strongly inhibited the release of beta-glucuronidase and lysozyme from rat neutrophils stimulated with formyl-Met-Leu-Phe/cytochalasin B (fMLP/CB). Compound 1 strongly inhibited superoxide anion formation in fMLP/CB or phorbol 12-myristate 13-acetate (PMA)-stimulated rat neutrophils. Compound 1 exhibited potent inhibitory effect on tumor-necrosis factor-alpha ( TNF-alpha) formation in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells while 1 and 4 showed potent inhibitory effects on TNF-alpha formation in LPS/IFN-gamma (interferon-gamma)-stimulated murine microglial cell lines N9.

Congenital generalized lipodystrophy in a 4-month-old infant.

Congenital generalized lipodystrophy (CGL, Berardinelli-Seip syndrome) is a rare autosomal recessive disorder with a clinical presentation of paucity of adipose tissue, muscular hypertrophy, organomegaly, and insulin-resistant diabetes. A 4-month-old Taiwanese female infant had hepatosplenomegaly and low body weight gain despite a voracious appetite. Hypermetabolism, hyperhidrosis, loss of subcutaneous fat, muscular hypertrophy, acanthosis nigricans, hypertrichosis, and marked hypertriglyceridemia were also noted. Liver histology revealed fatty change and portal-to-portal bridging fibrosis. Clinical features, serum biochemistry, and liver histology were compatible with the diagnosis of CGL. She was given a special diet characterized by calorie restriction and partial substitution of long-chain triglycerides with medium-chain triglycerides. The serum triglyceride concentration subsequently decreased. This present case suggests that extensive fatty infiltration and subsequent cirrhosis of the liver may be the earliest complication of CGL.

International trends and patterns of primary liver cancer.

Primary liver cancer (PLC) is common in many areas of the developing world, but uncommon in most of the developed world. Some evidence suggests, however, that the global pattern of PLC may be changing. To clarify this issue, we examined incidence rates for PLC over the 15-year time period, 1978-92, in selected cancer registries around the world. With some exceptions, developed countries have experienced PLC increases in incidence whereas developing countries have experienced declines. Although the reasons for the trends are not entirely clear, the increased seroprevalence of HCV in the developed world and the elimination of HBV-cofactors in the developing world are likely to have contributed to the patterns. Further progress against PLC may be seen in the developing world once the HBV-vaccinated segment of the population reaches adulthood. Published 2001 Wiley-Liss, Inc.

Two phenanthraquinones from Dendrobium moniliforme.

Two phenanthraquinones were isolated from the stems of Dendrobium moniliforme. They were identified by interpretation of spectral data as 2,6-dimethoxy-1,4,5,8-phenanthradiquinone and 5-hydroxy-3,7-dimethoxy-1,4-phenanthraquinone, named moniliformin (1) and denbinobin (2), respectively. This is the first report of compound 1, which possesses a novel 1,4,5,8-diquinone moiety in the phenanthraquinone skeleton. Compound 2 showed potent antiinflammatory effects in vitro.

Methamphetamine potentiates ischemia/reperfusion insults after transient middle cerebral artery ligation.

BACKGROUND AND PURPOSE: Previous studies have indicated that both methamphetamine (MA) and ischemia/reperfusion injuries involve reactive oxygen species formation and activation of apoptotic mechanism. That MA could have a synergistic or additive effect with stroke-induced brain damage is possible. The purpose of the present study was to investigate whether administration of MA in vivo would potentiate ischemic brain injury. METHODS: Adult CD-1 mice were pretreated with MA or saline. Each animal later was anesthetized with chloral hydrate and placed in a stereotaxic frame. A subset of animals received intracerebral administration of glial cell line-derived neurotrophic factor (GDNF). The right middle cerebral artery and bilateral carotids were transiently occluded for 45 minutes. Regional cerebral blood flow was measured by laser Doppler. Animals were sacrificed for triphenyltetrazolium chloride staining and p53 mRNA Northern blot assay after 24 hours of reperfusion. Cortical and striatal GDNF levels were assayed by ELISA. RESULTS: We found that pretreatment with MA increased ischemia-induced cerebral infarction. Ischemia or MA alone enhanced p53 mRNA expression. Moreover, MA potentiated expression of p53 mRNA in the ischemic mouse brain. MA pretreatment decreased GDNF levels in ischemic striatum. Intracerebral administration of GDNF before ischemia reduced MA-facilitated infarction. CONCLUSIONS: Our data indicate that MA exacerbates ischemic insults in brain, perhaps through the inhibition of GDNF-mediated pathways and suggest that MA may antagonize endogenous neuroprotective pathways as part of its mechanism of action.

Regulation of nitric oxide synthase 2 in rabbit corneal cells.

PURPOSE: The purpose of these studies was to investigate the role of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin 1beta (IL-1beta), and transforming growth factor-beta (TGF-beta) in the regulation of inducible nitric oxide synthase (NOS2) activity in rabbit corneal cells. METHODS: Rabbit corneal epithelial, stromal, and endothelial cells were grown in culture and treated with cytokines and growth factors, alone or in combination. NOS activity was measured at times up to 72 hours after treatment by assaying the culture medium for nitrite using the Griess reaction. Cell lysates were analyzed by Western blot analysis for NOS2 protein. RNA was isolated and amplified with NOS1-, NOS2-, and NOS3-specific primers by RT-PCR. RESULTS: NOS2 expression was induced by combined cytokine treatment from nondetectable levels to abundant levels in low passage (<4) stromal cells and to low levels in corneal endothelial cells but not in corneal epithelial cells. In the absence of IFN-gamma, little or no nitrite accumulation was induced by TNF-alpha, IL-1beta, or lipopolysaccharide (LPS) treatment. The inductive effects of IFN-gamma were antagonized in a dose-dependent manner by the myxoma virus rabbit IFN-gamma receptor homolog, M-T7. rRaIFN-gamma, in combination with IL-1beta and TNF-alpha, induced the appearance of NOS2 mRNA within 24 hours but detectable nitrite did not accumulate in large amounts (>10 microM) until after 24 hours postinduction. NOS2 was identified as a 130 kDa protein on Western blot analysis using monoclonal antibody against murine NOS2. TGF-beta(1) and beta(2) inhibited the accumulation of cytokine-induced nitrite in a dose-dependent manner while not significantly reducing the steady state level of NOS2 mRNA. The activity of the induced NOS was inhibited by 1400W, a NOS2-selective inhibitor, but not 7-nitroindazole, a NOS1-selective inhibitor. CONCLUSIONS: In cultured corneal stromal cells, NOS2 expression was upregulated by IFN-gamma in combination with IL-1beta and TNF-alpha but not by any of these cytokines alone, while TGF-beta downregulated the activity. Cultures of corneal epithelial cells could not be induced to express NOS2, yet cultures of endothelial cells produced low amounts of NO in response to cytokines. The NOS1 and NOS3 isoforms were not detected in any of these corneal cells.