NKG2D defines tumor-reacting effector CD8+ T cells within tumor microenvironment.

For successful immunotherapy for cancer, it is important to understand the immunological status of tumor antigen-specific CD8+ T cells in the tumor microenvironment during tumor progression. In this study, we monitored the behavior of B16OVA-Luc cells in mice immunized with a model tumor antigen ovalbumin (OVA). Using bioluminescence imaging, we identified the time series of OVA-specific CD8+ T-cell responses during tumor progression: initial progression, immune control, and the escape phase. As a result of analyzing the status of tumor antigen-specific CD8+ cells in those 3 different phases, we found that the expression of NKG2D defines tumor-reacting effector CD8+ T cells. NKG2D may control the fate and TOX expression of tumor-reacting CD8+ T cells, considering that NKG2D blockade in OVA-vaccinated mice delayed the growth of the B16OVA-Luc2 tumor and increased the presence of tumor-infiltrating OVA-specific CD8+ T cells.

Separating host and microbiome contributions to drug pharmacokinetics and toxicity.

The gut microbiota is implicated in the metabolism of many medical drugs, with consequences for interpersonal variation in drug efficacy and toxicity. However, quantifying microbial contributions to drug metabolism is challenging, particularly in cases where host and microbiome perform the same metabolic transformation. We combined gut commensal genetics with gnotobiotics to measure brivudine drug metabolism across tissues in mice that vary in a single microbiome-encoded enzyme. Informed by these measurements, we built a pharmacokinetic model that quantitatively predicts microbiome contributions to systemic drug and metabolite exposure, as a function of bioavailability, host and microbial drug-metabolizing activity, drug and metabolite absorption, and intestinal transit kinetics. Clonazepam studies illustrate how this approach disentangles microbiome contributions to metabolism of drugs subject to multiple metabolic routes and transformations.

ProTides of BVdU as potential anticancer agents upon efficient intracellular delivery of their activated metabolites.

Nucleosides represent a major chemotherapeutic class for treating cancer, however their limitations in terms of cellular uptake, nucleoside kinase-mediated activation and catabolism are well-documented. The monophosphate pro-nucleotides known as ProTides represents a powerful strategy for bypassing the dependence on active transport and nucleoside kinase-mediated activation. Herein, we report the structural tuning of BVdU ProTides. Forty six phosphoramidates were prepared and biologically evaluated against three different cancer cell lines; murine leukemia (L1210), human CD4+ T-lymphocyte (CEM) and human cervical carcinoma (HeLa). Twenty-fold potency enhancement compared to BVdU was achieved against L1210 cells. Interestingly, a number of ProTides showed low micromolar activity against CEM and HeLa cells compared to the inactive parent BVdU. The ProTides showed poor, if any measurable toxicity to non-tumourigenic human lung fibroblast cell cultures. Separation of four pairs of the diastereoisomeric mixtures and comparison of their spectral properties, biological activities and enzymatic activation rate is reported.

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization.

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell synchronization can be difficult for many cell systems. Standard practice is to block cell cycle progression at a specific stage and then release the accumulated cells producing a wave of cells progressing through the cycle in unison. However, some cell types find this block toxic resulting in abnormal cell cycling, or even mass death. Bromodeoxyuridine (BrdU) uptake can be used to track the cell cycle stage of individual cells. Cells incorporate this synthetic thymidine analog, while synthesizing new DNA during S phase. By providing BrdU for a brief period it is possible to mark a pool of cells that were in S phase while the BrdU was present. These cells can then be tracked through the remainder of the cell cycle and into the next round of replication, permitting the duration of the cell cycle phases to be determined without the need to induce a potentially toxic cell cycle block. It is also possible to determine and correlate the expression of both internal and external proteins during subsequent stages of the cell cycle. These can be used to further refine the assignment of cell cycle stage or assess effects on other cellular functions such as checkpoint activation or cell death.

Study on Interaction between 5-Bromo-4-thio-2'-deoxyuridine and human serum albumin by spectroscopy and molecular docking.

The interaction between 5-Bromo-4-thio-2'-deoxyuridine (4-SBrdU) and human serum albumin (HSA) was investigated by the methods of UV-vis absorbance, fluorescence and circular dichroism (CD) spectroscopy and molecular docking under simulative physiological conditions. The results showed that the quenching mechanism of HAS by 4-SBrdU was dynamic fluorescence quenching, hydrophobic interaction was the main intermolecular force based on thermodynamic data, the fluorescence experimental results were in agreement with results obtained by the molecular docking study.

Recolonization of Mutans Streptococci after Application of Chlorhexidine Gel

Streptococcus mutans is specifically suppressed by intensive treatment with chlorhexidine gel, but the time for recolonization and the effect on other oral bacteria are not totally clear. In this study, recolonization of mutans streptococci was evaluated in nine healthy adult volunteers, who were highly colonized with this microorganism. Stimulated saliva was collected before (baseline) and at 1, 7, 14, 21 and 28 days after application of 1% chlorhexidine gel on volunteers' teeth for two consecutive days. On each day, the gel was applied using disposable trays for 3 x 5 min with intervals of 5 min between each application. Saliva was plated on blood agar to determine total microorganisms (TM); on mitis salivarius agar to determine total streptococci (TS) and on mitis salivarius agar plus bacitracin to determine mutans streptococci (MS). Chlorhexidine was capable of reducing the counts of MS and the proportion of MS with regard to total microorganisms (%MS/TM) (p<0.05), but these values did not differ statistically from baseline (p>0.05) after 14 days for MS and 21 days for %MS/TM. The counts of TM and TS and the proportion of MS to total streptococci did not differ statistically from baseline (p>0.05) after chlorhexidine treatment. The results suggest that the effect of chlorhexidine gel treatment on suppression of mutans streptococci is limited to less than a month in highly colonized individuals.

Streptococcus mutans é especificamente suprimido pelo tratamento intensivo com clorexidina em gel, mas o tempo de recolonização e o efeito em outras bactérias orais não está totalmente claro. Nesse estudo, a recolonização de estreptococos do grupo mutans foi avaliado em nove voluntários adultos saudáveis, os quais eram altamente colonizados por esse microrganismo. Saliva estimulada foi coletada antes (baseline) e 1, 7, 14, 21 e 28 dias após a aplicação de clorexidina em gel a 1% nos dentes dos voluntários por dois dias consecutivos. Em cada dia, o gel foi aplicado utilizando moldeiras descartáveis por 3 x 5 min com intervalos de 5 min entre cada aplicação. A saliva foi inoculada em ágar sangue para determinação dos microrganismos totais (MT); em mitis salivarius ágar para determinação dos estreptococos totais (ET) e em meio mitis salivarius com bacitracina para determinar a contagem de estreptococos do grupo mutans (EGM). O tratamento com clorexidina foi capaz de reduzir as contagens de EGM e a proporção de EGM em relação aos microrganismos totais (%EGM/MT) (p<0,05), mas esses valores não diferiram estatisticamente do baseline (p>0,05) após 14 dias para EGM e 21 dias para %EGM/MT. As contagens de MT e ET e a proporção de EGM em relação a estreptococos totais não difereriram estatisticamente do baseline (p>0,05) após o tratamento com clorexidina. Os resultados sugerem que o efeito do tratamento com clorexidina em gel na supressão de estreptococos do grupo mutans é limitado a menos de um mês em indivíduos altamente colonizados. .

Identification of adult stem cells in Schwalbe's line region of the primate eye.

PURPOSE: To identify stem cells in the chamber angle of the monkey eye by detection of 5-bromo-2'-deoxyuridine (BrdU) long-term retention. METHODS: Four cynomolgus monkeys were treated with BrdU via subcutaneous pumps for 4 weeks. The eyes of two animals were processed immediately thereafter (group 1) while in the other animals, BrdU treatment was discontinued for 4 weeks to allow identification of cells with long-term BrdU retention (group 2). The number of BrdU-positive nuclei was quantified, and the cells were characterized by immunohistochemistry and transmission electron microscopy (TEM). RESULTS: The number of BrdU-positive cells was higher at Schwalbe's line covering the peripheral end of Descemet's membrane than in Schlemm's canal (SC) endothelium, trabecular meshwork (TM), and scleral spur (SS). Labeling with BrdU in SC, TM, and SS was less intense and the number of labeled cells was smaller in group 2 than in group 1. In contrast, in cells of Schwalbe's line the intensity of BrdU staining and the number of BrdU-positive cells was similar when group 1 and 2 monkeys were compared with each other, indicating long-term BrdU retention. Cells that were BrdU-positive in Schwalbe's line region stained for the stem cell marker OCT4. Details of a stem cell niche in Schwalbe's line region were identified by TEM. CONCLUSIONS: We provide evidence for a niche in the Schwalbe's line region harboring cells with long-term BrdU retention and OCT4 immunoreactivity. The cells likely constitute a population of adult stem cells with the capability to compensate for the loss of TM and/or corneal endothelial cells.

Flow cytometric determination of 5-bromo-2'-deoxyuridine pharmacokinetics in blood serum after intraperitoneal administration to rats and mice.

5-Bromo-2'-deoxyuridine (BrdU) is a marker that is widely used to label S-phase cells in neurobiological research in most common doses 50 or 100 mg/kg per single intraperitoneal (i.p.) injection. However, the important data regarding its pharmacokinetics in rodents are still missing. The aim of our study was to investigate the BrdU level in serum after a single i.p. injection to adult rats (doses: 50 or 100 mg/kg) and adult mice (50 mg/kg). The animals were killed at selected time-points after the BrdU injection, and proliferating tumour cells (cell lines HCT-116 and HL-60) were co-cultivated with isolated blood sera. BrdU incorporated in the DNA of the S-phase tumour cells was stained with an anti-BrdU antibody and analysed using flow cytometry. In rats, the efficacies of BrdU labelling of S-phase cells in both in vitro and in vivo conditions were compared in the 50 and 100 mg/kg groups. According to our results, BrdU was in saturated concentration to label almost all S-phase cells for 60 min in both doses and was detectable in blood serum until 120 min after the single i.p. injection. However, the 100 mg/kg dose of BrdU did not provide a prolonged staining period to offset the potentially higher toxicity in comparison with the 50 mg/kg dose. In mice, due to their faster metabolism, the concentration of BrdU in blood serum was sufficient to label the whole population of S-phase cells for only 15 min after the i.p. injection, then dropped rapidly.

S phase cell percentage normalized BrdU incorporation rate, a new parameter for determining S arrest.

In this study, a new parameter, S phase cell percentage (S fraction) normalized BrdU (SFN-BrdU) incorporation rate, was introduced to detect S arrest. The results showed a positive linear correlation between the BrdU incorporation rate and the S fraction in unperturbed 16HBE cells. Theoretical analysis indicated that only S arrest could result in a decrease in the SFN-BrdU incorporation rate. Additionally, the decrease in SFN-BrdU incorporation rate and the activation of DNA damage checkpoints further demonstrated that S arrest was induced by diethyl sulfate treatment of 16HBE cells. In conclusion, SFN-BrdU incorporation rate can be used to detecting S arrest.

Bromodeoxyuridine (BrdU)-label-retaining cells in mouse terminal bronchioles.

Adult male mice were continuously treated with bromodeoxyuridine (BrdU) for 1, 2, or 4 weeks by an osmotic pump. To detect BrdU-label-retaining cells (LRCs), putative progenitor/stem cells, other animals were continuously treated with BrdU for 2 weeks, and were then kept without any treatments for 2, 6, or 18 months. The lungs were fixed with 4% paraformaldehyde, and were paraffin-embedded. We observed terminal bronchioles with BrdU immunostaining alone or with BrdU immunostaining accompanying immunostaining for Clara cell secretory protein (CCSP), forkhead box protein J1 (FoxJ1), or calcitonin gene-related peptide (CGRP). The average incidences of BrdU-incorporated cells in the terminal bronchioles after 1, 2, and 4 weeks of continuous BrdU infusion were 6.2%, 11.9%, and 23.1%, respectively. Most BrdU-incorporated cells in these periods were CCSP-immunoreactive (91.7%, 91.3%, and 88.2%, respectively), which means progenitor function of Clara cells. FoxJ1-immunoreactive BrdU-incorporated cells were fewer (5.4%, 3.0%, 2.7%, respectively). The average incidences of BrdU-LRCs in the terminal bronchioles after 2, 6, and 18 months were 7.2%, 4.3, and 2.7%, respectively. Most BrdU-LRCs were CCSP-immunoreactive (91.0%, 92.7%, and 89.6%, respectively), and FoxJ1-immunoreactive BrdU-LRCs were fewer (6.0%, 5.7%, and 2.1%, respectively). CGRP-positive BrdU-incorporated cells were occasional. CGRP-positive BrdU-LRCs were detected in 17.6% of neuroepithelial bodies (NEBs) at 2 months, but disappeared at 6 months. BrdU-positive stem cell candidates, which locate at the brochiolo-alveolar duct junction or cover NEB, were few throughout this study. In conclusion, in the lungs treated only with BrdU, CCSP-immunoreactive cells are important to maintain homeostasis in the terminal bronchiolar epithelium.

Enhanced radiosensitization by the cationic liposome-encapsulated thymidine analogue BrdU through the increased intracellular BrdU-uptake on human melanoma as compared to anionic or nonionic liposomal or free BrdU.

The synthetic thymidine analogue, 5-bromo-2'-deoxy-uridine (BrdU) was encapsulated in cationic liposome composed of dipalmitoylphosphatidylcholine, cholesterol and stearylamine (molar ratio = 1/1/0.2; diameter = 120 nm), and the radiosensitization of cationic liposomal BrdU was assessed on human melanoma cells HMV-II, with comparing to anionic or nonionic liposomal BrdU and free-BrdU. HMV-II cells were pretreated by cationic liposomal BrdU or free-BrdU and then exposed to X-ray, followed by WST-8 assay to examine cell proliferation. The radiation-induced change of nuclei was defined with Hoechst33342 staining. The rates of thymidine replacement by BrdU and DNA double-strand breaks on HMV-II cells were determined with an anti-BrdU antibody and anti-53BP1 antibody, respectively. On 7th day after X-ray irradiation at 3 Gy, cell proliferation was suppressed more markedly in the administration of cationic liposomal BrdU than free-BrdU at equivalent BrdU doses. Giant polykaryocytes were observed in cationic liposomal BrdU-treated HMV-II cells. Radiosensitization was enhanced dose-dependently along with BrdU doses of 0.1-0.8 µM in the order: cationic liposomal BrdU > anionic liposomal BrdU > nonionic liposomal BrdU (see symbol) free-BrdU. Similarly, the cationic liposomal BrdU augmented the rate of thymidine-moiety replacement by BrdU and DNA double-strand breaks more appreciably than free-BrdU. Therefore, the cationic liposome-encapsulation of BrdU would be one of favorable drug deliveries for facilitating the X-ray therapy against cancer.

A mechanistic model for bromodeoxyuridine dilution naturally explains labelling data of self-renewing T cell populations.

Bromodeoxyuridine (BrdU) is widely used in immunology to detect cell division, and several mathematical models have been proposed to estimate proliferation and death rates of lymphocytes from BrdU labelling and de-labelling curves. One problem in interpreting BrdU data is explaining the de-labelling curves. Because shortly after label withdrawal, BrdU+ cells are expected to divide into BrdU+ daughter cells, one would expect a flat down-slope. As for many cell types, the fraction of BrdU+ cells decreases during de-labelling, previous mathematical models had to make debatable assumptions to be able to account for the data. We develop a mechanistic model tracking the number of divisions that each cell has undergone in the presence and absence of BrdU, and allow cells to accumulate and dilute their BrdU content. From the same mechanistic model, one can naturally derive expressions for the mean BrdU content (MBC) of all cells, or the MBC of the BrdU+ subset, which is related to the mean fluorescence intensity of BrdU that can be measured in experiments. The model is extended to include subpopulations with different rates of division and death (i.e. kinetic heterogeneity). We fit the extended model to previously published BrdU data from memory T lymphocytes in simian immunodeficiency virus-infected and uninfected macaques, and find that the model describes the data with at least the same quality as previous models. Because the same model predicts a modest decline in the MBC of BrdU+ cells, which is consistent with experimental observations, BrdU dilution seems a natural explanation for the observed down-slopes in self-renewing populations.

Increased 5-bromo-2'-deoxyuridine incorporation in various brain structures following passive avoidance training in mice.

We studied the effects of training on DNA synthesis intensity in mouse brain. Brain cells where DNA synthesis-associated processes took place under the influence of training were detected by immunohistochemical labeling of DNA molecules with synthetic thymine analogue 5-bromo-2'-deoxyuridine. The number of 5-bromo-2'-deoxyuridine-positive cell increased in various structures of the brain under the influence of training.

LINE-1 retrotransposon copies are amplified during murine early embryo development.

Two large families of retrotransposons, that is, LINE-1 (Long Interspersed Nuclear Elements-1) and endogenous retroviruses, encode reverse transcriptase (RT) proteins in vertebrates. We previously showed that mouse preimplantation embryos are endowed with an endogenous, functional RT activity. Inhibiting that activity by microinjecting antisense oligonucleotides against a highly active LINE-1 family member in mouse oocytes blocked developmental progression between the two- and four-blastomere stages, indicating that LINE-1-encoded RT activity is strictly required at this critical transition in early development. Here we show that incubation of mouse zygotes with 5'-bromodeoxyuridine (BrdU) yields massive incorporation of this nucleoside analogue in newly synthesized DNA; surprisingly, a significant incorporation still occurs in both zygotic pronuclei in the presence of aphidicolin, a specific inhibitor of DNA replication. This aphidicolin-resistant BrdU incorporation is quantitatively abolished when embryos are simultaneously exposed to abacavir, a nucleoside RT inhibitor, indicating its retrotranscription-dependent nature. Moreover, quantitative PCR analysis revealed a burst of new synthesis of LINE-1 copies at the zygote- and two-cell embryo stages. These findings support the conclusion that RT-dependent amplification of LINE-1 retrotransposons is a distinctive feature of early embryonic genomes. Its physiological involvement in preimplantation murine development is discussed.

Detection of BrdU-label retaining cells in the lacrimal gland: implications for tissue repair.

The purpose of the present study was to determine if the lacrimal gland contains 5-bromo-2'-deoxyuridine (BrdU)-label retaining cells and if they are involved in tissue repair. Animals were pulsed daily with BrdU injections for 7 consecutive days. After a chase period of 2, 4, or 12 weeks, the animals were sacrificed and the lacrimal glands were removed and processed for BrdU immunostaining. In another series of experiments, the lacrimal glands of 12-week chased animals were either left untreated or were injected with interleukin 1 (IL-1) to induce injury. Two and half days post-injection, the lacrimal glands were removed and processed for BrdU immunostaining. After 2 and 4 weeks of chase period, a substantial number of lacrimal gland cells were BrdU(+) (11.98 ± 1.84 and 7.95 ± 1.83 BrdU(+) cells/mm(2), respectively). After 12 weeks of chase, there was a 97% decline in the number of BrdU(+) cells (0.38 ± 0.06 BrdU(+) cells/mm(2)), suggesting that these BrdU-label retaining cells may represent slow-cycling adult stem/progenitor cells. In support of this hypothesis, the number of BrdU labeled cells increased over 7-fold during repair of the lacrimal gland (control: 0.41 ± 0.09 BrdU(+) cells/mm(2); injured: 2.91 ± 0.62 BrdU(+) cells/mm(2)). Furthermore, during repair, among BrdU(+) cells 58.2 ± 3.6 % were acinar cells, 26.4 ± 4.1% were myoepithelial cells, 0.4 ± 0.4% were ductal cells and 15.0 ± 3.0% were stromal cells. We conclude that the murine lacrimal gland contains BrdU-label retaining cells that are mobilized following injury to generate acinar, myoepithelial and ductal cells.

Gender-related differences in repopulation and early tumor response to preoperative radiotherapy in rectal cancer patients.

PURPOSE: Inhibition of tumor proliferation rate based on bromodeoxyuridine labelling index (BrdUrdLI), S-phase fraction (SPF) and MIB-1 labelling index (MIB-1 LI) as an early rectal cancer response to preoperative radiotherapy (RT). METHODS AND MATERIALS: A total of 122 patients qualified either for short RT (5 Gy/fraction/5 days) and surgery about 1 week after RT (schedule I) or for short RT and a 4-week interval before surgery (schedule II). Tumor samples were taken twice from each patient: before RT and at the time of surgery. In each sample, the BrdUrdLI, SPF and MIB-1 were calculated. Early tumor response was assessed by a biologist, a pathologist and surgeons. RESULTS: Fifty-six patients were treated according to schedule I and 66 patients according to schedule II. Mean BrdUrdLI, SPF and MIB-1 LI before RT were 8.8%, 21.0% and 53.3%, respectively, and these values did not differ between the two compared groups. After RT, tumors showed statistically significant growth inhibition based on all assessed biological markers. As pretreatment assessed parameter was not predictive for early clinical and pathologic tumor response, prognostic role of the relative value (RV), that is, the ratio of assessed parameter after RT to before RT for each of the assessed markers, was considered. The ratios were calculated separately for fast and slowly proliferating tumors and separately for male and female patients. Fast proliferating tumors were more responsive. Differences with regard to sex were visible only in slowly proliferating tumors. Accelerated cell repopulation (4.8-28%/day) was noticed in female slowly proliferating tumors about 4 weeks after RT. Only for relative MIB-1 LI it was possible to show significant correlation with pathological tumor regression. Lack of such correlation for BrdUrdLI and SPF might reflect accelerated repopulation, particularly in slowly proliferating female tumors. CONCLUSIONS: Accelerated repopulation was noticed in slowly proliferating tumors in females about 4 weeks after RT.

ß-Cell replication and islet neogenesis following partial pancreatectomy.

Partial pancreatectomy is one of the most commonly used models in the study of ß-cell regeneration. The mechanism by which regeneration occurs in this model has been controversial, with some claiming that islet and ß-cell neogenesis is important, while others claim that ß-cell replication is predominant. Here, we combined a time course analysis with continuous BrdU administration to study ß-cell regeneration following partial pancreatectomy. While exocrine cells in regenerating areas were highly proliferative and positive for BrdU, islets in regenerating areas were negative for BrdU one week after partial pancreatectomy, suggesting that they were derived from preexisting islets rather than being neogenic. The insulin-positive cells in ducts that have been reported by others and taken as evidence of ß-cell neogenesis were present in regenerating regions of the pancreas, but were relatively uncommon and were not highly proliferative, suggesting that they could not account for significant islet neogenesis. Consistent with a lack of islet neogenesis, regenerating areas following a second partial pancreatectomy were devoid of islets. ß-cell replication was detectable at a high frequency two weeks following partial pancreatectomy and was present at a similar frequency in both regenerating and preexisting regions of the pancreas. In summary, our data indicate that islet neogenesis following partial pancreatectomy does not occur.

Hypoxia predicts aggressive growth and spontaneous metastasis formation from orthotopically grown primary xenografts of human pancreatic cancer.

Hypoxia in solid tumors is associated with treatment resistance and increased metastatic potential. Although hypoxia has been reported in pancreatic cancer patients, there is little direct evidence that this contributes to their overall poor prognosis. To address this, we examined the associations between hypoxia and biological aggression in a series of patient-derived xenografts grown orthotopically. Early passage xenografts were established from 16 patients undergoing surgery for pancreatic cancer and maintained in the pancreas of immune-deprived mice. Hypoxic cells were labeled using the 2-nitroimidazole probe EF5 and stained for immunofluorescence microscopy of tissue sections or as cell suspensions for flow cytometry. Bromodeoxyuridine (BrdUrd) uptake, microvessel density, cleaved caspase-3, and the differentiation markers E-cadherin, cytokeratin 19, and vimentin were analyzed in relation to hypoxia. Orthotopic implants closely resembled the histology of the original surgical samples. The 16 primary xenografts showed a wide range in their growth rates and metastatic potential, reminiscent of the spectrum of behavior seen in the clinic. EF5 labeling, tumor growth rates, and metastatic patterns were highly consistent within replicates, indicating a significant transmissible (genetic or epigenetic) component. Hypoxia was highly correlated with rapid tumor growth, increased BrdUrd uptake, and with spontaneous metastasis formation. mRNA expression analysis showed increased expression of genes involved in cell survival and proliferation in the hypoxic models. The results suggest that hypoxia is a major adverse prognostic factor in pancreatic cancer patients and support the introduction of techniques to measure hypoxia directly in patients and the development of treatment protocols to target hypoxia.

Deafferentation enhances neurogenesis in the young and middle aged hippocampus but not in the aged hippocampus.

Increased neurogenesis in the dentate gyrus (DG) after brain insults such as excitotoxic lesions, seizures, or stroke is a well known phenomenon in the young hippocampus. This plasticity reflects an innate compensatory response of neural stem cells (NSCs) in the young hippocampus to preserve function or minimize damage after injury. However, injuries to the middle-aged and aged hippocampi elicit either no or dampened neurogenesis response, which could be due to an altered plasticity of NSCs and/or the hippocampus with age. We examined whether the plasticity of NSCs to increase neurogenesis in response to a milder injury such as partial deafferentation is preserved during aging. We quantified DG neurogenesis in the hippocampus of young, middle-aged, and aged F344 rats after partial deafferentation. A partial deafferentation of the left hippocampus without any apparent cell loss was induced via administration of Kainic acid (0.5 µg in 1.0 µl) into the right lateral ventricle of the brain. In this model, degeneration of CA3 pyramidal neurons and dentate hilar neurons in the right hippocampus results in loss of commissural axons which leads to partial deafferentation of the dendrites of dentate granule cells and CA1-CA3 pyramidal neurons in the left hippocampus. Quantification of newly born cells that are added to the dentate granule cell layer at postdeafferentation days 4-15 using 5'-bromodeoxyuridine (BrdU) labeling revealed greatly increased addition of newly born cells (∼three fold increase) in the deafferented young and middle-aged hippocampi but not in the deafferented aged hippocampus. Measurement of newly born neurons using doublecortin (DCX) immunostaining also revealed similar findings. Analyses using BrdU-DCX dual immunofluorescence demonstrated no changes in neuronal fate-choice decision of newly born cells after deafferentation, in comparison to the age-matched naive hippocampus in all age groups. Thus, the plasticity of hippocampal NSCs to increase DG neurogenesis in response to a milder injury such as partial hippocampal deafferentation is preserved until middle age but lost at old age.

Significant human beta-cell turnover is limited to the first three decades of life as determined by in vivo thymidine analog incorporation and radiocarbon dating.

AIMS: Diabetes mellitus results from an absolute or relative deficiency of insulin-producing pancreatic ß-cells. The turnover rate of adult human ß-cells remains unknown. We employed two techniques to examine adult human islet ß-cell turnover and longevity in vivo. METHODS: Subjects enrolled in National Institutes of Health clinical trials received thymidine analogs [iododeoxyuridine (IdU) or bromodeoxyuridine (BrdU)] 8 d to 4 yr prior to death. Archival autopsy samples from 10 patients (aged 17-74 yr) were employed to assess ß-cell turnover by scoring nuclear analog labeling within insulin-staining cells. Human adult ß-cell longevity was determined by estimating the cells' genomic DNA integration of atmospheric (14)C. DNA was purified from pancreatic islets isolated from cadaveric donors; whole islet prep DNA was obtained from a 15-yr-old donor, and purified ß-cell DNA was obtained from two donors (ages 48 and 80 yr). (14)C levels were then determined using accelerator mass spectrometry. Cellular "birth date" was determined by comparing the subject's DNA (14)C content relative to a well-established (14)C atmospheric prevalence curve. RESULTS: In the two subjects less than 20 yr of age, 1-2% of the ß-cell nuclei costained for BrdU/IdU. No ß-cell nuclei costained in the eight patients more than 30 yr old. Consistent with the BrdU/IdU turnover data, ß-cell DNA (14)C content indicated that the "birth date" of cells occurred within the subject's first 30 yr of life. CONCLUSIONS: Under typical circumstances, human ß-cells and their cellular precursors are established by young adulthood.