Targeting PD-1 in CD8+ T Cells with a Biomimetic Bilirubin-5-fluoro-2-deoxyuridine-Bovine Serum Albumin Nanoconstruct for Effective Chemotherapy against Experimental Lymphoma.

We fabricated bilirubin-bovine serum albumin (BR-BSA) nanocomplexes as candidates for the delivery of 5-fluoro-2-deoxyuridine (5FUdr) against experimental murine lymphoma. BR was attached to 5FUdr via acid-labile ester bonds mimicking small-molecule drug conjugates. The construct was self-assembled with BSA through strong noncovalent interactions with high drug occupancy in the core and labeled with folic acid (FA) to target cancer cells. The BR-5FUdr-BSA-FA nanoconstruct exhibits excellent biocompatibility, prevents nephrotoxicity, and is tolerated by red blood cells and mononuclear cells. The construct also showed increased accumulation in lymph nodes and tumor cells. BR-5FUdr-BSA-FA caused prolonged growth inhibition and apoptosis, enhanced mitochondrial reactive oxygen species generation, and minimized the viability of parental and doxorubicin-resistant Dalton's lymphoma cells. Treatment of tumor-bearing mice with BR-5FUdr-BSA-FA significantly increased the life span of the animals, improved their histopathological parameters, and downregulated PD-1 expression, suggesting the potential of the construct for 5FUdr delivery to treat lymphoma.

Discovery of 2-aminoisobutyric acid ethyl ester (AIBEE) phosphoramidate prodrugs for delivering nucleoside HCV NS5B polymerase inhibitors.

Our HCV research program investigated novel 2'-dihalogenated nucleoside HCV polymerase inhibitors and identified compound 1, a 5'-phosphoramidate prodrug of 2'-deoxy-2'-α-bromo-ß-chloro uridine. Although 1 had a favorable in vitro activity profile in HCV replicons, oral dosing in dog resulted in low levels of the active 5'-triphosphate (TP) in liver. Metabolism studies using human hepatocytes provided a simple assay for screening alternative phosphoramidate prodrug analogs. Compounds that produced high TP concentrations in hepatocytes were tested in dog liver biopsy studies. This method identified 2-aminoisobutyric acid ethyl ester (AIBEE) phosphoramidate prodrug 14, which provided 100-fold higher TP concentrations in dog liver in comparison to 1 (4 and 24 h after 5 mg/kg oral dose).

LC-MS/MS assay for the quantitation of FdCyd and its metabolites FdUrd and FU in human plasma.

The hypomethylating agent 5-fluoro-2'-deoxycytidine (FdCyd, NSC 48006) is being evaluated clinically both via the intravenous route and via the oral route in combination with 3,4,5,6-tetrahydrouridine (THU), a potent inhibitor of FdCyd catabolism. To determine the pharmacokinetics of FdCyd and downstream metabolites, we developed and validated an LC-MS/MS assay for the quantitation of FdCyd, 5-fluoro-2'-deoxyuridine (FdUrd), and 5-fluorouracil (FU) in 0.2mL human plasma. After acetonitrile protein precipitation, the sample was split and separate chromatography was achieved for FdCyd with a Synergi Polar-RP column and for FdUrd and FU with a Shodex Asahipak NH2P-50 2D column. Gradients of 0.1% acetic acid in acetonitrile and water were used. Detection with a Quattromicro quadrupole mass spectrometer with electrospray ionization in positive-ion (FdCyd) or negative-ion (FdUrd and FU) multiple reaction monitoring (MRM) mode. The assay was linear from 5 to 3000ng/mL for all three analytes and proved to be accurate (96.7-105.5%) and precise (<8.1%CV), and fulfilled FDA criteria for bioanalytical method validation. We demonstrated the suitability of this assay for measuring FdCyd and metabolites FdUrd and FU in plasma from a patient who was administered 120mg PO FdCyd 30min after 3000mg THU. Our LC-MS/MS assay will be an essential tool to further define the pharmacology of FdCyd in ongoing and future studies.

Discovery of an Orally Active and Liver-Targeted Prodrug of 5-Fluoro-2'-Deoxyuridine for the Treatment of Hepatocellular Carcinoma.

We report a series of novel O-(substituted benzyl) phosphoramidate prodrugs of 5-fluoro-2'-deoxyuridine for the treatment of hepatocellular carcinoma. Through structure optimization, the o-methylbenzyl analog (1t) was identified as an orally bioavailable and liver-targeted lead compound. This lead prodrug is well-tolerated at a dose up to 3 g/kg in Kuming mice via oral administration. An efficacy study demonstrated that it possesses good inhibitory effect (61.67% and 72.50%, respectively) on tumor growth in a mouse xenograft model. A metabolism study in Sprague-Dawley rats suggested that 1t can release the desired 5'-monophosphate in the liver with high liver-targeting index.

Exploring the intracellular pharmacokinetics of the 5-fluorouracil nucleotides during capecitabine treatment.

AIM: Three intracellularly formed metabolites are responsible for the antineoplastic effect of capecitabine: 5-fluorouridine 5'-triphosphate (FUTP), 5-fluoro-2'-deoxyuridine 5'-triphosphate (FdUTP), and 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). The objective of this study was to explore the pharmacokinetics of these intracellular metabolites during capecitabine treatment. METHODS: Serial plasma and peripheral blood mononuclear cell (PBMC) samples were collected from 13 patients treated with capecitabine 1000 mg QD (group A) and eight patients receiving capecitabine 850 mg m(-2) BID for fourteen days, every three weeks (group B). Samples were collected on day 1 and, for four patients of group B, also on day 14. The capecitabine and 5-fluorouracil (5-FU) plasma concentrations and intracellular metabolite concentrations were determined using LC-MS/MS. Pharmacokinetic parameters were estimated using non-compartmental analysis. RESULTS: Only FUTP could be measured in the PBMC samples. The FdUTP and FdUMP concentrations were below the detection limits (LOD). No significant correlation was found between the plasma 5-FU and intracellular FUTP exposure. The FUTP concentration-time profiles demonstrated considerable inter-individual variation and accumulation of the metabolite in PBMCs. FUTP levels ranged between

Phase I dose escalation and pharmacokinetic evaluation of two different schedules of LY2334737, an oral gemcitabine prodrug, in patients with advanced solid tumors.

BACKGROUND: This Phase-I-study aimed to determine the recommended Phase-II-dosing-schedule of LY2334737, an oral gemcitabine prodrug, in patients with advanced/metastatic solid tumors. Pharmacokinetics, cytokeratin-18 (CK18) levels, genetic polymorphisms, and antitumor activity were additionally evaluated. METHODS: Patients received escalating doses of LY2334737 either every other day for 21 days (d) followed by 7 days-drug-free period (QoD-arm) or once daily for 7 days every other week (QD-arm). The 28 days-cycles were repeated until disease progression or unacceptable toxicity. Standard 3 + 3 dose-escalation was succeeded by a dose-confirmation phase (12 additional patients to be enrolled on the maximum tolerated dose [MTD]). RESULTS: Forty-one patients received QoD- (40-100 mg) and 32 QD-dosing (40-90 mg). On QoD, 3/9 patients experienced dose-limiting toxicities (DLTs) on the 100 mg dose (2 × G3 diarrhea, 1 × G3 transaminase increase); 1 additional DLT (G3 diarrhea) occurred during dose confirmation at 90 mg (12 patients). On QD, 1 patient each experienced DLTs on 60 mg (G3 transaminase increase) and 80 mg (G3 prolonged QTcF-interval); 2/7 patients had 3 DLTs on the 90 mg dose (diarrhea, edema, liver-failure; all G3). The MTD was established at 90 mg for the QoD-arm. Seven patients on QoD and 4 on QD achieved SD (no CR + PR). Pharmacokinetics showed a dose-proportional increase in exposure of LY2334737 and dFdC without accumulation after repeated dosing. Significant increases in CK18 levels were observed. Genetic polymorphism of the cytidine deaminase gene (rs818202) could be associated with ≥ G3 hepatotoxicity. CONCLUSIONS: Both schedules displayed linear pharmacokinetics and acceptable safety profiles. The recommended dose and schedule of LY2334737 for subsequent Phase-II-studies is 90 mg given QoD for 21 day.

Colitis induces enteric neurogenesis through a 5-HT4-dependent mechanism.

BACKGROUND: The intestine is known to contain enteric neuronal progenitors, but their precise identity and the mechanisms that activate them remain unknown. Based on the evidence for the neurogenic role of serotonin (5-HT) in the postnatal gut and the observation of enteric neuronal hyperplasia in inflammatory bowel disease, we hypothesized that colitis induces a neurogenic response through 5-HT4 receptor signaling. METHODS: We examined the effects of 5-HT4 agonism on colonic neurogenesis and gliogenesis in vitro and in vivo in adult mice using dextran sodium sulfate to experimentally induce colitis. RESULTS: In vitro, 5-HT4 agonism led to increased neuronal proliferation and density. Induction of experimental colitis in vivo similarly resulted in increased numbers of myenteric neurons, and this was inhibited by 5-HT4 antagonism. Interestingly, both in vitro and in vivo, 5-HT4 signaling increased glial cell proliferation but did not increase glial cell numbers, leading us to hypothesize that glia may give rise to neurons. After induction of colitis in normal, Nestin-GFP and Sox2-GFP transgenic mice, it was revealed that multiple glial markers (Sox2, Nestin, and CD49b) became strongly expressed by enteric neurons. Immunoselected enteric glia were found to give rise to neurons in culture, and this was inhibited in the presence of 5-HT4 blockade. Finally, isolated glia gave rise to a neuronal network upon transplantation into aganglionic embryonic avian hindgut. CONCLUSIONS: These results show that colitis promotes enteric neurogenesis in the adult colon through a serotonin-dependent mechanism that drives glial cells to transdifferentiate into neurons.

Phase 1b study of the oral gemcitabine 'Pro-drug' LY2334737 in combination with capecitabine in patients with advanced solid tumors.

Background This Phase 1b study aimed to determine the recommended Phase 2 dose of LY2334737, an oral pro-drug of gemcitabine, in combination with capecitabine, an oral pro-drug of 5-fluorouracil, in patients with advanced solid tumors. In addition, pharmacokinetics (PK) and tumor response were evaluated. Patients and methods Patients with advanced/metastatic solid tumors received 650 mg/m(2) capecitabine twice daily (BID) and escalating doses of LY2334737 once daily (QD; initial dose 10 mg/day), both for 14 days followed by 7-day drug holiday. Cycles were repeated until progressive disease (PD) or unacceptable toxicity. Results Fifteen patients received a median of 2 (range 1-7) treatment cycles; 14 patients discontinued due to PD, 1 due to toxicity (pyrexia). LY2334737 doses up to 40 mg/day were explored. Three dose-limiting toxicities were reported by 2 patients (fatigue, diarrhea, hyponatremia; all Grade 3). Seven patients achieved stable disease. Enrollment was stopped after unexpected hepatic toxicities were observed with LY2334737 QD in a study of Japanese patients. PK parameters for LY2334737 were consistent with the first-in-human study of LY2334737; PK data after 14 day combination treatment revealed no drug-drug interactions between LY2334737 and capecitabine. Conclusions No drug interactions or unexpected toxicities were observed in US patients when LY2334737 at doses up to 40 mg/day was administered QD in combination with capecitabine BID; the maximum tolerated dose was not reached.

Phase 1 dose escalation and pharmacokinetic evaluation of oral gemcitabine prodrug (LY2334737) in combination with docetaxel in patients with advanced solid tumors.

PURPOSE: This Phase 1 study aimed to determine the recommended Phase 2 dose of LY2334737, an oral gemcitabine prodrug, when combined with standard dose docetaxel treatment in patients with advanced solid tumors. Pharmacokinetics (PK) and antitumor activity were additionally evaluated. METHODS: Patients with advanced/metastatic solid tumors received escalating doses of LY2334737 once daily (QD) for 14 days, followed by a 7-day drug-free period. Docetaxel was given at 75 mg/m(2) every 3 weeks (q3w). Cycles were repeated until progressive disease (PD) or unacceptable toxicity. RESULTS: Of 22 patients recruited, all Caucasian, 7 received an LY2334737 dose of 10 mg/day, 10 received 20 mg/day, 5 received 30 mg/day. Nineteen patients discontinued due to PD, 2 due to adverse events, 1 due to investigator decision. Dose-limiting toxicities: 2× febrile neutropenia (G3), 2× fatigue (1× G2, 1× G3), 1× neutropenia (G4). The maximum tolerated dose (MTD) was identified to be 10 mg/day. Two patients achieved partial response, 10 patients stable disease. Enrollment was stopped after unexpected hepatic toxicities were observed with LY2334737 QD for 14 days per cycle in another study of Japanese patients. PK data were consistent with the first-in-man study of LY2334737 and did not reveal any drug-drug interaction between LY2334737 and docetaxel. CONCLUSIONS: Combination of LY2334737 at doses up to 30 mg/day QD for 14 days per cycle with docetaxel 75 mg/m(2) q3w resulted in an undesirable toxicity profile and a low MTD of 10 mg/day. Alternative treatment schedules of LY2334737 should be explored.

Evaluation of (131/123)I-5-iodo-2'-deoxycytidine as a novel proliferation probe in a tumor mouse model.

This study evaluated a radioiodinated deoxycytidine analog, (131)I-5-iodo-2'-deoxycytidine ([(131)I]ICdR), as a novel proliferation probe and compared it with (131)I-5-iodo-2'-deoxyuridine ([(131)I]IUdR) in a NG4TL4 sarcoma-bearing mouse model. As an imaging agent, the biological characteristics of [(123)I]IUdR is not satisfactory due to its metabolic instability and short biological half-life in vivo. With [(123)I]ICdR/SPECT it was possible to clearly delineate the tumor lesion at 1h post-injection (tumor-to-muscle ratio 7.74) in tumor-bearing mice. The results of biodistribution were consistent with those observed in scintigraphic imaging. This study demonstrated that [(131)I]ICdR is a more promising SPECT probe than [(131)I]IUdR for imaging proliferation.

Phase I study of oral gemcitabine prodrug (LY2334737) in Japanese patients with advanced solid tumors.

PURPOSE: LY2334737 is an oral gemcitabine prodrug. This Phase I study assessed the safety and tolerability of LY2334737 in Japanese patients with solid tumors and evaluated pharmacokinetics (PK), pharmacodynamics, and antitumor activity. METHODS: Patients with advanced/metastatic solid tumors received escalating doses of LY2334737 once daily for 14 days, followed by a 7-day drug-free period. Cycles were repeated until discontinuation criteria were met. RESULTS: Of 13 patients treated, 3 received 20 mg/day, 6 received 30 mg/day, 4 received 40 mg/day. On the 40 mg dose, 3 patients experienced dose-limiting toxicities (DLTs): hepatic toxicities (e.g., Grade [G]3/4 transaminase and G1-3 bilirubin elevation) and G4 thrombocytopenia; all 3 showed features of disseminated intravascular coagulation. One additional DLT occurred on the 30 mg dose (G3 transaminase elevation). Exploratory pharmacogenetic analyses identified a genetic variation in the CES2 gene potentially associated with these DLTs. PK data showed no clear relationship between the AUC of gemcitabine and its incorporation into leukocyte DNA; 2 of the 3 DLT patients had high incorporation. Two patients (30 mg/day) achieved stable disease with progression-free survival lasting 135 and 155 days. CONCLUSIONS: LY2334737 was tolerated by Japanese patients up to 30 mg/day. The toxicities observed at the 40 mg dose may require the development of alternative dosing schedules.

The effects of the 4-(4-Methylpiperazine)phenyl group on nucleosides and oligonucleotides: cellular delivery, detection, and stability.

As drug candidates, one promising way to improve the cellular delivery efficacy of oligonucleotides is to introduce a cationic group. By introducing a cationic moiety into the oligonucleotide structure, they become capable of approaching the cell surface and also of crossing the cellular membrane. In an effort to develop cell-permeable oligonucleotides, we examined the piperazinephenyl-bearing 2'-deoxyuridine ((PP)U), which can be not only cationic but also fluorescent as a cationic monomer for cationic oligonucleotides. Several modified DNA oligonucleotides with different numbers of (PP)U building blocks were synthesized and evaluated for the effect on thermal stability and conformation by the introduction of (PP)U. The cellular delivery of modified oligonucleotides was different depending on the number of (PP)U building blocks. Furthermore, these (PP)U-modified oligonucleotides had sufficient fluorescence that we were able to identify the delivery results without the use of conventional fluorescent tags. They were predominantly localized in the cell cytoplasm. In addition, they were stable enough after 3 hours in the presence of nuclease. These results showed that a piperazinephenyl moiety that is conjugated with nucleobase is able to deliver and detect the oligonucleotides, which suggests that this concept of 'dual-function oligonucleotides' might be utilized in diagnostics, therapeutics, and as a convenient biological tool for probing the activity of oligonucleotides inside cells.

Radiolabeled 5-iodo-3'-O-(17beta-succinyl-5alpha-androstan-3-one)-2'-deoxyuridine and its 5'-monophosphate for imaging and therapy of androgen receptor-positive cancers: synthesis and biological evaluation.

High levels of androgen receptor (AR) are often indicative of recurrent, advanced, or metastatic cancers. These conditions are also characterized by a high proliferative fraction. 5-Radioiodo-3'-O-(17beta-succinyl-5alpha-androstan-3-one)-2'-deoxyuridine 8 and 5-radioiodo-3'-O-(17beta-succinyl-5alpha-androstan-3-one)-2'-deoxyuridin-5'-yl monophosphate 13 target AR. They are also degraded intracellularly to 5-radioiodo-2'-deoxyuridine 1 and its monophosphate 20, respectively, which can participate in the DNA synthesis. Both drugs were prepared at the no-carrier-added level. Precursors and methods are readily adaptable to radiolabeling with various radiohalides suitable for SPECT and PET imaging, as well as endoradiotherapy. In vitro and in vivo studies confirm the AR-dependent interactions. Both drugs bind to sex hormone binding globulin. This binding significantly improves their stability in serum. Biodistribution and imaging studies show preferential uptake and retention of 8 and 13 in ip xenografts of human ovarian adenocarcinoma cells NIH:OVCAR-3, which overexpress AR. When these drugs are administered at therapeutic dose levels, a significant tumor growth arrest is observed.

Preferential tumor targeting and selective tumor cell cytotoxicity of 5-[131/125I]iodo-4'-thio-2'-deoxyuridine.

PURPOSE: Auger electron emitting radiopharmaceuticals are attractive for targeted nanoirradiation therapy, provided that DNA of malignant cells is selectively addressed. Here, we examine 5-[123/125/131I]iodo-4'-thio-2'-deoxyuridine (ITdU) for targeting DNA in tumor cells in a HL60 xenograft severe combined immunodeficient mouse model. EXPERIMENTAL DESIGN: Thymidine kinase and phosphorylase assays were done to determine phosphorylation and glycosidic bond cleavage of ITdU, respectively. The biodistribution and DNA incorporation of ITdU were determined in severe combined immunodeficient mice bearing HL60 xenografts receiving pretreatment with 5-fluoro-2'-deoxyuridine (FdUrd). Organ tissues were dissected 0.5, 4, and 24 h after radioinjection and uptake of [131I]ITdU (%ID/g tissue) was determined. Cellular distribution of [125I]ITdU was imaged by microautoradiography. Apoptosis and expression of the proliferation marker Ki-67 were determined by immunohistologic staining using corresponding paraffin tissue sections. RESULTS: ITdU is phosphorylated by thymidine kinase 1 and stable toward thymidylate phosphatase-mediated glycosidic bond cleavage. Thymidylate synthase-mediated deiodination of [123/125/131I]ITdU was inhibited with FdUrd. Pretreatment with FdUrd increased preferentially tumor uptake of ITdU resulting in favorable tumor-to-normal tissue ratios and tumor selectivity. ITdU was exclusively localized within the nucleus and incorporated into DNA. In FdUrd-pretreated animals, we found in more than 90% of tumor cells apoptosis induction 24 h postinjection of ITdU, indicating a highly radiotoxic effect in tumor cells but not in cells of major proliferating tissues. CONCLUSION: ITdU preferentially targets DNA in proliferating tumor cells and leads to apoptosis provided that the thymidylate synthase is inhibited.

Synthesis and biodistribution of 3'-fluoro-5-[(131)I]iodo-2'-deoxyuridine: a comparative study of [(131)I]FLIdU and [(18)F]FLT.

The radioiodinated 3'-fluorothymidine (FLT) analogue 3'-fluoro-5-[(131)I]iodo-2'-deoxyuridine ([(131)I]FLIdU) was synthesized, with iodine mimicking the methyl group of pyrimidine. [(131)I]FLIdU was accessible by direct electrophilic iodination using Iodogen as oxidant. Optimized amounts of the oxidant allowed radiochemical yields of about 70% after a reaction time of 10 min in an aqueous buffer medium at 90 degrees C. The uptake of [(131)I]FLIdU in a DoHH2 leukemia xenograft mouse model and in healthy mice revealed moderate FLIdU accumulation, followed by a significant washout of activity in proliferating tissues such as splenic and tumor tissues. In contrast, intraperitoneal coinjection with [(18)F]FLT showed high uptake and high activity retention up to 2 h, in both splenic and tumor tissues. Uptake in stomach tissues and increasing fractions of [(131)I]iodide in urine indicated metabolic instability of [(131)I]FLIdU due to rapid deiodination. Therefore, [(131)I]FLIdU alone does not seem to be a promising compound, neither for diagnostic imaging nor for potential therapeutic applications.

Rapid quantitation of plasma 2'-deoxyuridine by high-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry and its application to pharmacodynamic studies in cancer patients.

A novel method employing high-performance liquid chromatograph-mass spectrometry (LC-MS) has been developed and validated for the quantitation of plasma 2'-deoxyuridine (UdR). It involves a plasma clean-up step with strong anion-exchange solid-phase extraction (SAX-SPE) followed by HPLC separation and atmospheric pressure chemical ionization mass spectrometry detection (APCI-MS) in a selected-ion monitoring (SIM) mode. The ionization conditions were optimised in negative ion mode to give the best intensity of the dominant formate adduct [M+HCOO]- at m/z 273. Retention times were 7.5 and 12.5 min for 2'-deoxyuridine and 5-iodo-2'-deoxyuridine, an iodinated analogue internal standard (IS), respectively. Peak area ratios of 2'-deoxyuridine to IS were used for regression analysis of the calibration curve. The latter was linear from 5 to 400 nmol/l using 1 ml sample volume of plasma. The average recovery was 81.5% and 78.6% for 2'-deoxyuridine and 5-iodo-deoxyuridine, respectively. The method provides sufficient sensitivity, precision, accuracy and selectivity for routine analysis of human plasma 2'-deoxyuridine concentration with the lowest limit of quantitation (LLOQ) of 5 nmol/l. Clinical studies in cancer patients treated with the new fluoropyrimidine analogue capecitabine (N4-pentoxycarbonyl-5'-5-fluorocytidine) have shown that plasma 2'-deoxyuridine was significantly elevated after 1 week of treatment, consistent with inhibition of thymidylate synthase (TS). These findings suggest that the mechanism of antiproliferative toxicity of capecitabine is at least partly due to TS inhibitory activity of its active metabolite 5-fluoro-2'-deoxyuridine monophosphate (FdUMP). Monitoring of plasma UdR concentrations have the potential to help clinicians to guide scheduling of capecitabine or other TS inhibitors in clinical trials. Marked differences of plasma 2'-deoxyuridine between human and rodents have also been confirmed. In conclusion, the LC-MS method developed is simple, highly selective and sensitive and permits pharmacodynamic studies of TS inhibitors in several species.

Proximal airway mucous cells of ovalbumin-sensitized and -challenged Brown Norway rats accumulate the neuropeptide calcitonin gene-related peptide.

Mucous cell hypersecretion and increased neuropeptide production play a role in the exacerbation of symptoms associated with asthma. The source of these neuropeptides have been confined to the contributions of small afferent nerves or possibly neuroendocrine cells. We tested the hypothesis that repeated exposure to allergen would alter the sources and abundance of neuropeptides in airways. Right middle lobes from rats (8 wk old) exposed to 2.5% ovalbumin (OVA) for five episodes (30 min each) or filtered air were inflation fixed with paraformaldehyde. The lobes were dissected to expose the airway tree, permeabilized with DMSO, and incubated in antibody to rat calcitonin gene-related peptide (CGRP), followed with a fluorochrome-labeled second antibody. CGRP-positive structures were imaged via confocal microscopy. Airways were later embedded in plastic and sectioned for cell identification. In animals challenged with OVA, CGRP-positive cells, not neuroendocrine or neuronal in origin (confirmed by a lack of protein gene product 9.5 signal), were recorded along the axial path. In section, this fluorescent signal was localized to granules within epithelial cells. Alcian blue/periodic acid-Schiff staining of these same sections positively identify these cells as mucous cells. Mucous cells of animals not challenged with OVA were not positive for CGRP. We conclude that episodic allergen exposure results in the accumulation of CGRP within mucous cells, creating a new source for the release of this neuropeptide within the airway.

Neural stem cell detection, characterization, and age-related changes in the subventricular zone of mice.

The mammalian brain contains neural stem cells (NSCs) that allow continued neurogenesis throughout the life of the animal. However, neurogenesis is known to decline during aging and, to the extent that neurogenesis is required for normal CNS function, this may contribute to neurodegenerative disease. Decreased neurogenesis could result from loss of NSCs or dysfunction at some later step, and distinguishing these possibilities is important for understanding the cause of the decline. However, because of the inability to distinguish NSCs from their rapidly dividing progeny in situ, it has not been possible to quantitatively assess the NSC populations in young and old animals. In this report we show that the G1 phase-specific expression of the replication factor Mcm2 is a useful marker for detecting slowly cycling putative NSCs in situ and confirm the identity of these cells using both cytosine beta-D-arabinofuranoside (Ara-C) treatment and a double nucleoside analog-labeling technique. The ability to distinguish NSCs from proliferative progenitors has allowed characterization of the expression of several markers including Nestin, Musashi, and GFAP in these different cell types. Furthermore, comparison of the NSC populations in the subventricular zones of young (2-4 months) and old (24-26 months) mice demonstrates an approximately twofold reduction in the older mice. A similar twofold reduction is also observed in the number of neurospheres recovered in culture from old relative to young animals. The reduction in the neural stem cell population documented here is sufficient to account for the reduced level of neurogenesis in old animals.