MiR129-5p-loaded exosomes suppress seizure-associated neurodegeneration in status epilepticus model mice by inhibiting HMGB1/TLR4-mediated neuroinflammation.

BACKGROUND: Neuroinflammation contributes to both epileptogenesis and the associated neurodegeneration, so regulation of inflammatory signaling is a potential strategy for suppressing epilepsy development and pathological progression. Exosomes are enriched in microRNAs (miRNAs), considered as vital communication tools between cells, which have been proven as potential therapeutic method for neurological disease. Here, we investigated the role of miR129-5p-loaded mesenchymal stem cell (MSC)-derived exosomes in status epilepticus (SE) mice model. METHODS: Mice were divided into four groups: untreated control (CON group), kainic acid (KA)-induced SE groups (KA group), control exosome injection (KA + Exo-con group), miR129-5p-loaded exosome injection (KA + Exo-miR129-5p group). Hippocampal expression levels of miR129-5p, HMGB1, and TLR4 were compared among groups. Nissl and Fluoro-jade B staining were conducted to evaluate neuronal damage. In addition, immunofluorescence staining for IBA-1 and GFAP was performed to assess glial cell activation, and inflammatory factor content was determined by ELISA. Hippocampal neurogenesis was assessed by BrdU staining. RESULTS: The expression of HMGB1 was increased after KA-induced SE and peaking at 48 h, while hippocampal miR129-5p expression decreased in SE mice. Exo-miR129-5p injection reversed KA-induced upregulation of hippocampal HMGB1 and TLR4, alleviated neuronal damage in the hippocampal CA3, reduced IBA-1 + and GFAP + staining intensity, suppressed SE-associated increases in inflammatory factors, and decreased BrdU + cell number in dentate gyrus. CONCLUSIONS: Exosomes loaded with miR129-5p can protect neurons against SE-mediated degeneration by inhibiting the pro-inflammatory HMGB1/TLR4 signaling axis.

APOBEC3G rescues cells from the deleterious effects of DNA damage.

Human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G (hA3G), a member of the APOBEC family, was described as an anti-HIV-1 restriction factor, deaminating reverse transcripts of the HIV-1 genome. Several types of cancer cells that express high levels of A3G, such as diffuse large B-cell lymphoma cells and glioblastomas, show enhanced cell survival after ionizing radiation and chemotherapy treatments. Previously, we showed that hA3G promotes (DNA) double-strand breaks repair in cultured cells and rescues transgenic mice from a lethal dose of ionizing radiation. Here, we show that A3G rescues cells from the detrimental effects of DNA damage induced by ultraviolet irradiation and by combined bromodeoxyuridine and ultraviolet treatments. The combined treatments stimulate the synthesis of cellular proteins, which are exclusively associated with A3G expression. These proteins participate mainly in nucleotide excision repair and homologous recombination DNA repair pathways. Our results implicate A3G inhibition as a potential strategy for increasing tumor cell sensitivity to genotoxic treatments.

A life-threatening drug-drug interaction between capecitabine and brivudine in a patient with metastatic breast cancer.

In the current report we present the case of a patient experiencing a life-threatening drug-drug interaction involving the concurrent administration of capecitabine and brivudine. A 65- year-old female with metastatic breast cancer was commenced on brivudine for Herpes Zoster, while on capecitabine treatment, by a physician unfamiliar with the potential repercussions of this drug-drug interaction. As a result, she developed skin rash, severe oral mucositis, and severe and prolonged pancytopenia. These side effects were attributed to a serious interaction of capecitabine with brivudine, resulting in inhibition of dihydropyrimidine dehydrogenase. The patient was admitted for supportive care including intravenous hydration, parenteral nutrition, mouth care solutions, fluconazole, antimicrobial therapy, filgrastim, red blood cell and platelet transfusions. She successfully recovered and was discharged on the 26th day after her admission. Drug-drug interactions can be serious, even life-threatening; thus the physicians should be cautious when prescribing new drugs.

Characterization of the blastogenic response to LPS of bovine peripheral blood mononuclear cells.

Mitogens are diverse compounds of plant and microbial origin, widely employed to test immunocompetence in animals. The blastogenic response of bovine Peripheral Blood Mononuclear Cells (PBMC) to lypopolysaccharides (LPS) has been investigated in our laboratories for a long time. In particular, a possible correlation between blastogenic response to LPS and disease resistance of periparturient dairy cows had been observed in previous studies. Most important, low responder cows presented a higher frequency of disease cases after calving, compared with high responder animals. Owing to the above, different aspects of the blastogenic response to LPS were investigated on PBMC of healthy Friesian cows, using a 72-hour Bromodeoxyuridin (BrDU) cell proliferation assay. Stimulation with LPS induced little if any replication of bovine PBMC over 72 hours despite consistent BrDU detection in all the PBMC samples under study. Poor replication of LPS-stimulated PBMC was confirmed by cell cycle and cell growth flow cytometry analyses. In particular, LPS stimulation gave rise to very low percentages of S phase cells, sometimes lower than in control, unstimulated cells, as opposed to Concanavalin A-stimulated PBMC. Magnetic separation and analysis of BrDU-treated bovine PBMC after exposure to LPS showed that both B and CD4 T cells are involved in the blastogenic response to LPS, in contrast with current data based on human and murine models. Finally, LPS caused an early, specific up-regulation of TNF-α and TLR4 genes in bovine PBMC, and significant correlations were shown between the expression of inflammatory cytokine and Indoleamine-pyrrole 2,3-dioxygenase (IDO1) genes. On the whole, our data indicate that differences in the blastogenic response to LPS could be partly accounted for by heterogenicity of responding cells (B and T lymphocytes), which might also have an impact on induction and regulation of inflammatory responses and endotoxin tolerance.

Repeated exposure to 5-bromo-2'-deoxyuridine causes decreased proliferation and low-grade inflammation in the lungs of mice.

Incorporation of 5-bromo-2'-deoxyuridine (BrdU) into proliferating cells has been used to label dividing cells in many tissues. Although BrdU has been shown to be genotoxic, teratogenic and mutagenic, such adverse effects have largely been ignored by researchers. We determined whether long-term BrdU exposure causes any histopathological changes in the lungs of mice. Eight-week-old male C57/BL6J mice were administered BrdU by intraperitoneal injection on 3 consecutive days of each week for 14 weeks. While no obvious structural changes such as tissue damage, fibrosis, emphysema, airway remodeling, vascular thickening or tumorigenesis were noted, a moderate degree of macrophage infiltration was observed in the airways and lung parenchyma in the lungs of the mice exposed repeatedly to BrdU (BrdU-exposed mice). The proliferative activities of the airway and alveolar epithelial and mesenchymal cells were reduced in the BrdU-exposed mice, although the numbers of these cells in the lungs were maintained. Double immunofluorescence study of the lungs of the BrdU-exposed mice showed overexpression of IL-6 in the airway epithelial and alveolar wall cells, some of which were also double-positive for BrdU. These results indicate that long-term exposure to BrdU inhibits cell proliferation and induces low-grade inflammation in the lungs of mice. Our findings underscore the need for caution in the interpretation of studies that involve long-term exposure to BrdU.

Brivudin induced delirium: a case report.

Brivudin is an oral antiviral agent used to treat herpes zoster infections. Common side effects of brivudin include nausea and headache. This report describes delirium in a patient who used brivudin for herpes zoster treatment, which consequently remitted after drug cessation on the fourth day of medication use. To our knowledge, no such side effect has been reported to date. However, it is important that clinicians who prescribe brivudin are aware that treatment cessation is likely to result in total clinical recovery.

BrdU birth dating can produce errors in cell fate specification in chick brain development.

Birth dating neurons with bromodeoxyuridine (BrdU) labeling is an established method widely employed by neurobiologists to study cell proliferation in embryonic, postnatal, and adult brain. Birth dating studies in the chick dorsal telencephalon and the mammalian striatum have suggested that these structures develop in a strikingly similar manner, in which neurons with the same birth date aggregate to form "isochronic clusters." Here we show that isochronic cluster formation in the chick dorsal telencephalon is an artifact. In embryos given standardly employed doses of BrdU, we observed isochronic clusters but found that clusters were absent with BrdU doses close to the limits of detection. In addition, in situ hybridization experiments established that neurons in the clusters display errors in cell type specification: BrdU cell clusters in nidopallium adopted a mesopallial neuronal fate, mesopallial clusters were misspecified as nidopallial cells, and in some instances, the BrdU clusters failed to express neuronal differentiation markers characteristic of the dorsal telencephalon. These results demonstrate that the chick dorsal telencephalon does not develop by isochronic cluster formation and highlight the need to test the integrity of BrdU-treated tissue with gene expression markers of regional and cell type identity.

Antivirals for management of herpes zoster including ophthalmicus: a systematic review of high-quality randomized controlled trials.

BACKGROUND: There is lack of consensus from randomized controlled trials on the efficacy of antivirals in the management of herpes zoster. Therefore, a systematic review and meta-analysis was undertaken to provide better understanding of effectiveness of antivirals in management of herpes zoster. METHODS: A total of 12 randomized controlled trials with 7,277 patients were included in the review. Trials compared one antiviral to another (aciclovir, valaciclovir, famciclovir or brivudin) for a minimum of 7 days in immunocompetent patients presenting with herpes zoster diagnosed within 72 h of symptom onset. Primary outcome was reduction in pain. RESULTS: Compared with aciclovir, valaciclovir showed significant reduction in herpes-zoster-associated pain up to 112 days. The largest risk reduction in pain (36%) was seen at 21-30 days (relative risk [RR] 0.64, 95% CI 0.59, 0.70) with number needed to treat to benefit (NNT) of 3 (95% CI 2.7, 3.8). Famciclovir was also superior to aciclovir with a 46% reduction in risk of pain at 28-30 days (RR 0.54, 95% CI 0.48, 0.68) with NNT of 3 (95% CI 2, 5). Time to lesion healing and adverse effect profile was comparable. CONCLUSIONS: Evidence from quality trials have shown significant reduction in risk of pain with valaciclovir and famciclovir for management of herpes zoster including ophthalmicus. Valaciclovir or famciclovir should be preferred treatment options in patients with herpes zoster as they both provide significant reduction in risk of herpes-zoster-associated pain. Furthermore, the superior pharmacokinetics and more convenient dosing regimens with the use of valaciclovir and famciclovir clearly make them the preferred treatment option.

The effects of nitric oxide inhibition prior to kainic acid treatment on neuro- and gliogenesis in the rat dentate gyrus in vivo and in vitro.

Treatment with the nitric oxide synthase (NOS) inhibitor, L-NAME prior to the induction of seizures with kainic acid (KA) [L-NAME+KA] increases the expression of activity-dependent neuroprotective protein (ADNP) in cells in the subgranular zone (SGZ) of the rat dentate gyrus 3-days after seizure induction (Cosgrave et al., 2009). Using the incorporation of BrdU we found that this protocol [L-NAME+KA] stimulates neuro- and gliogenesis. By comparison, L-NAME or KA alone produced smaller effects. Doublecortin+ (BrdU negative) neuroblasts in the SGZ also significantly increased with L-NAME+KA treatment, suggesting that L-NAME+KA cause more cells to differentiate into neurons. L-NAME alone increased BrdU+ astrocytes in the hilus implying that NO inhibits stem cell differentiation into astrocytes and may also influence their migration. Although NOS inhibition increased cell proliferation in vivo and in vitro it disrupted cell clustering as revealed by ADNP immunoreactivity. In vitro KA treatment resulted in eccentric nuclei, reduced neurite extension and branching in neurons and retracted processes of glia cells, these changes were inhibited with prior treatment of L-NAME suggesting that KA-induced NO production affects cell morphology. Consequently, this data suggests an important role for NO in regulating stem cell proliferation and their fate in the SGZ.

A novel model to measure the regenerative potential of the peripheral nervous system after experimental immunological demyelination.

BACKGROUND: Immunological demyelination is a proposed strategy to improve nerve regeneration in the peripheral nervous system. To investigate the remyelinating potential of Schwann cells in vivo in the peripheral nervous system, the authors have reproduced and expanded upon a novel model of immunological demyelination in the adult rat sciatic nerve. The authors demonstrate (1) the peripheral nervous system's quantitative, regenerative response to immunological demyelination and (2) whether Schwann cells within a region of demyelination are induced to divide in the presence of demyelinated axons. METHODS: The sciatic nerves of female Sprague-Dawley rats were exposed and injected with demyelinating agent bilaterally. At 3 days (n = 3), 7 days (n = 3), and 14 days (n = 3), the animals were euthanized for histological evaluation. A second group of animals (n = 3) was similarly injected with demyelinating agent and then exposed to bromodeoxyuridine between 48 and 72 hours after the onset of demyelination. These animals were euthanized soon after the last injection of bromodeoxyuridine. The tissue was analyzed for Schwann cells (labeled with antibodies to S100) and bromodeoxyuridine assay. RESULTS: A single epineural injection of complement proteins plus antibodies to galactocerebroside resulted in demyelination followed by Schwann cell remyelination. At 3 days after injection, peripheral nerve demyelination and Schwann cell proliferation were evident. Maximum demyelination was seen at 7 days; however, Schwann cell proliferation and remyelination peaked at 14 days after injection. CONCLUSIONS: These studies demonstrate an immunological model of demyelination and remyelination in the peripheral nervous system and quantitatively measure regenerative potential. This model will be used to isolate nerve segments and to measure their regenerative potential when given demyelinating agent after acute contusion and transection injuries.

BrdU immunohistochemistry for studying adult neurogenesis: paradigms, pitfalls, limitations, and validation.

Bromodeoxyuridine (BrdU) is a thymidine analog that incorporates DNA of dividing cells during the S-phase of the cell cycle. As such, BrdU is used for birth dating and monitoring cell proliferation. BrdU immunohistochemistry has been instrumental for the study of the development of the nervous system, and to confirm that neurogenesis occurs in the adult mammalian brain, including in human. However, the use of BrdU for studying neurogenesis is not without pitfalls and limitations. BrdU is a toxic and mutagenic substance. It triggers cell death, the formation of teratomas, alters DNA stability, lengthens the cell cycle, and has mitogenic, transcriptional and translational effects on cells that incorporate it. All of which have profound consequences on neurogenesis. BrdU is not a marker of the S-phase of the cell cycle. As a thymidine analog, it is a marker of DNA synthesis. Therefore, studying neurogenesis with BrdU requires distinguishing cell proliferation and neurogenesis from other events involving DNA synthesis, like DNA repair, abortive cell cycle reentry and gene duplication. BrdU labeling is currently the most used technique for studying adult neurogenesis in situ. However in many instances, appropriate controls have been overlooked and events reported as the generation of new neuronal cells in the adult brain misinterpreted, which makes BrdU labeling one of the most misused techniques in neuroscience.

Brivudin (bromovinyl deoxyuridine).

Brivudin is an oral thymidine analogue indicated for the early treatment of acute herpes zoster in immunocompetent adults. It has high, selective activity against varicella zoster virus (VZV), inhibiting VZV replication, possibly through competitive inhibition of viral DNA polymerase, or by acting as an alternative substrate to deoxythymidine triphosphate, causing viral DNA strand breakage. In a large, 7-day, phase III trial in immunocompetent patients with herpes zoster, once-daily brivudin 125mg was significantly more effective than oral acyclovir 800mg five times daily in reducing the mean time from start of treatment to last vesicular eruption, and was as effective as acyclovir at healing lesions and alleviating acute zoster-related pain. The likelihood of developing post-herpetic neuralgia (PHN) in immunocompetent patients aged > or =50 years was significantly lower with brivudin than with acyclovir. Brivudin was as effective as oral famciclovir 250mg three times daily in terms of the prevalence of PHN, the time to last vesicular eruption and lesion healing in another large, 7-day, phase III study in immunocompetent patients with herpes zoster. Oral brivudin is generally well tolerated, with a similar tolerability profile to those of oral acyclovir or famciclovir. Nausea was the most commonly reported adverse event.

Cumulative labeling of embryonic mouse neural retina with bromodeoxyuridine supplied by an osmotic minipump.

I describe a method for providing cumulative label of bromodeoxyuridine (BrdU) to mouse embryos. Commercially available osmotic pumps, which release their contents at a steady rate, were implanted subcutaneously in the interscapular space of pregnant mice on embryonic day (E) 9.5-12.5. Survival times varied from 4h to 37 days. Tissues (embryonic and neonatal eyes and maternal intestine) were immunochemically labeled for BrdU and examined histologically. The first detectably labeled cells appeared 4-7h post-implantation (hpi) and all cycling cells were labeled for at least 7 days post-implantation (dpi). Retinal development appeared normal. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) stained retinas that had been exposed to BrdU showed no more apoptotic cells than those unexposed. I conclude that the maternally implanted osmotic pump successfully provides cumulative BrdU labeling in the mouse embryo.