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Project description:Reprogramming of somatic cell nuclei to yield induced pluripotent stem (iPS) cells makes possible derivation of patient-specific stem cells for regenerative medicine. However, iPS cell generation is asynchronous and slow (2-3 weeks), the frequency is low (<0.1%), and DNA demethylation constitutes a bottleneck. To determine regulatory mechanisms involved in reprogramming, we generated interspecies heterokaryons (fused mouse embryonic stem (ES) cells and human fibroblasts) that induce reprogramming synchronously, frequently and fast. Here we show that reprogramming towards pluripotency in single heterokaryons is initiated without cell division or DNA replication, rapidly (1 day) and efficiently (70%). Short interfering RNA (siRNA)-mediated knockdown showed that activation-induced cytidine deaminase (AID, also known as AICDA) is required for promoter demethylation and induction of OCT4 (also known as POU5F1) and NANOG gene expression. AID protein bound silent methylated OCT4 and NANOG promoters in fibroblasts, but not active demethylated promoters in ES cells. These data provide new evidence that mammalian AID is required for active DNA demethylation and initiation of nuclear reprogramming towards pluripotency in human somatic cells.
| S-EPMC2906123 | biostudies-literature
Project description:In the brain, de novo gene expression driven by learning-associated neuronal activities is critical for the formation of long-term memories. However, the signaling machinery mediating neuronal activity-induced gene expression, especially the rapid transcription of immediate-early genes (IEGs) remains unclear. Cyclin-dependent kinases (Cdks) are a family of serine/threonine kinases that have been firmly established as key regulators of transcription processes underling coordinated cell cycle entry and sequential progression in nearly all types of proliferative cells. Cdk7 is a subunit of transcriptional initiation factor II-H (TFIIH) and the only known Cdk-activating kinase (CAK) in metazoans. Recent studies using a novel Cdk7 specific covalent inhibitor, THZ1, revealed important roles of Cdk7 in transcription regulation in cancer cells. However, whether Cdk7 plays a role in the regulation of transcription in neurons remains unknown. In this study, we present evidence demonstrating that, in post-mitotic neurons, Cdk7 activity is positively correlated with neuronal activities in cultured primary neurons, acute hippocampal slices and in the brain. Cdk7 inhibition by THZ1 significantly suppressed mRNA levels of IEGs, selectively impaired long-lasting synaptic plasticity induced by 4 trains of high frequency stimulation (HFS) and prevented the formation of long-term memories.
| S-EPMC5681959 | biostudies-literature
Project description:Humans with drug addiction exhibit compulsive drug-seeking associated with impairment of prefrontal cortex cognitive function. Whether prefrontal cortex dysfunction is a consequence of chronic drug exposure, or mediates the transition from drug use to drug dependence, is unknown. The current study investigates whether a history of escalated vs controlled cocaine intake is associated with specific working memory impairments, and long-lasting alterations of the dorsomedial prefrontal cortex and orbitofrontal cortex in rats. Working memory was assessed in rats with a history of extended (6 h per session) or limited (1 h per session) access to cocaine (0.5 mg/kg per injection), 3-17 days after the last self-administration session, using a delayed nonmatching-to-sample task. The density of neurons, oligodendrocytes, and astrocytes was quantified in the dorsomedial prefrontal cortex and orbitofrontal prefrontal cortex 2 months after the last self-administration session. Working memory impairments were observed after a history of chronic and escalated cocaine intake, but not after repeated limited access to cocaine. Moreover, working memory impairments were correlated with a decreased density of neurons and oligodendrocytes but not astrocytes in the dorsomedial prefrontal cortex, and with a decreased density of oligodendrocytes in the orbitofrontal cortex. Considering the role of the prefrontal cortex in goal-directed behavior, the prefrontal cortex dysfunctions observed here may exacerbate the loss of control associated with increased drug use and facilitate the progression to drug addiction.
| S-EPMC2760333 | biostudies-literature
Project description:Using FrCas(E) retrovirus-infected newborn mice as a model system, we have shown recently that a long-lasting antiviral immune response essential for healthy survival emerges after a short treatment with a neutralizing (667) IgG2a isotype monoclonal antibody (MAb). This suggested that the mobilization of adaptive immunity by administered MAbs is key for the success in the long term for the MAb-based passive immunotherapy of chronic viral infections. We have addressed here whether the anti-FrCas(E) protective endogenous immunity is the mere consequence of viral propagation blunting, which would simply give time to the immune system to react, and/or to actual immunomodulation by the MAb during the treatment. To this aim, we have compared viral replication, disease progression, and antiviral immune responses between different groups of infected mice: (i) mice treated with either the 667 MAb, its F(ab')(2) fragment, or an IgM (672) with epitopic specificity similar to that of 667 but displaying different effector functions, and (ii) mice receiving no treatment but infected with a low viral inoculum reproducing the initial viral expansion observed in their infected/667 MAb-treated counterparts. Our data show that the reduction of FrCas(E) propagation is insufficient on its own to induce protective immunity and support a direct immunomodulatory action of the 667 MAb. Interestingly, they also point to sequential actions of the administered MAb. In a first step, viral propagation is exclusively controlled by 667 neutralizing activity, and in a second one, this action is complemented by FcgammaR-binding-dependent mechanisms, which most likely combine infected cell cytolysis and the modulation of the antiviral endogenous immune response. Such complementary effects of administered MAbs must be taken into consideration for the improvement of future antiviral MAb-based immunotherapies.
| S-EPMC2937798 | biostudies-other
Project description:Transgenerational immune priming (TGIP) allows memory-like immune responses to be transmitted from parents to offspring in many invertebrates. Despite increasing evidence for TGIP in insects, the mechanisms involved in the transfer of information remain largely unknown. Here, we show that Drosophila melanogaster and Aedes aegypti transmit antiviral immunological memory to their progeny that lasts throughout generations. We observe that TGIP, which is virus and sequence specific but RNAi independent, is initiated by a single exposure to disparate RNA viruses and also by inoculation of a fragment of viral double-stranded RNA. The progeny, which inherit a viral DNA that is only a fragment of the viral RNA used to infect the parents, display enriched expression of genes related to chromatin and DNA binding. These findings represent a demonstration of TGIP for RNA viruses in invertebrates, broadly increasing our understanding of the immune response, host genome plasticity, and antiviral memory of the germline.
| S-EPMC7758158 | biostudies-literature
Project description:CD40/CD154 interactions are essential for productive antibody responses to T-dependent antigens. Memory CD4 T cells express accelerated helper functions and are less dependent on costimulation when compared with naïve T cells. Here, we report that donor-reactive memory CD4 T cells can deliver help to CD40-deficient B cells and induce high titers of IgG alloantibodies that contribute to heart allograft rejection in CD40-/- heart recipients. While cognate interactions between memory helper T and B cells are crucial for CD40-independent help, this process is not accompanied by germinal center formation and occurs despite inducible costimulatory blockade. Consistent with the extrafollicular nature of T/B cell interactions, CD40-independent help fails to maintain stable levels of serum alloantibody and induce differentiation of long-lived plasma cells and memory B cells. In summary, our data suggest that while CD40-independent help by memory CD4 T cells is sufficient to induce high levels of pathogenic alloantibody, it does not sustain long-lasting anti-donor humoral immunity and B cell memory responses. This information may guide the future use of CD40/CD154 targeting therapies in transplant recipients containing donor-reactive memory T cells.
| S-EPMC4019209 | biostudies-literature
Project description:Methamphetamine (METH) is a highly addictive and neurotoxic psychostimulant. Its use in humans is often associated with neurocognitive impairment. Whether this is due to long-term deficits in short-term memory and/or hippocampal plasticity remains unclear. Recently, we reported that METH increases baseline synaptic transmission and reduces LTP in an ex vivo preparation of the hippocampal CA1 region from young mice. In the current study, we tested the hypothesis that a repeated neurotoxic regimen of METH exposure in adolescent mice decreases hippocampal synaptic plasticity and produces a deficit in short-term memory. Contrary to our prediction, there was no change in the hippocampal plasticity or short-term memory when measured after 14 days of METH exposure. However, we found that at 7, 14, and 21 days of drug abstinence, METH-exposed mice exhibited a deficit in spatial memory, which was accompanied by a decrease in hippocampal plasticity. Our results support the interpretation that the deleterious cognitive consequences of neurotoxic levels of METH exposure may manifest and persist after drug abstinence. Therefore, therapeutic strategies should consider short-term as well as long-term consequences of methamphetamine exposure.
| S-EPMC3831527 | biostudies-literature
Project description:Visceral leishmaniasis caused by Leishmania donovani is the most severe systemic form of the disease. There are still no vaccines available for humans and there are limitations associated with the current therapeutic regimens for leishmaniasis. Recently, we reported functional importance of Arabino-1, 4-lactone oxidase (ALO) enzyme from L. donovani involved in ascorbate biosynthesis pathway. In this study, we have shown that ?ALO parasites do not affect the ability of null mutants to invade visceral organs but severely impair parasite persistence beyond 16 week in BALB/c mice and hence are safe as an immunogen. Both short term (5 week) and long term (20 week) immunization with ?ALO parasites conferred sustained protection against virulent challenge in BALB/c mice, activated splenocytes and resulted in induction of pro-inflammatory cytokine response. Protection in immunized mice after challenge correlated with the stimulation of IFN-? producing CD4(+) and CD8(+) T cells. Antigen-mediated cell immunity correlated with robust nitrite and superoxide generation, macrophage-derived oxidants critical in controlling Leishmania infection. Our data shows that live attenuated ?ALO parasites are safe, induce protective immunity and can provide sustained protection against Leishmania donovani. We further conclude that the parasites attenuated in their anti-oxidative defence mechanism can be exploited as vaccine candidates.
| S-EPMC4451804 | biostudies-other
Project description:Translating the recent success of chimeric antigen receptor (CAR) T cell therapy for hematological malignancies to solid tumors will necessitate overcoming several obstacles, including inefficient T cell tumor infiltration and insufficient functional persistence. Taking advantage of an orthotopic model that faithfully mimics human pleural malignancy, we evaluated two routes of administration of mesothelin-targeted T cells using the M28z CAR. We found that intrapleurally administered CAR T cells vastly outperformed systemically infused T cells, requiring 30-fold fewer M28z T cells to induce long-term complete remissions. After intrapleural T cell administration, prompt in vivo antigen-induced T cell activation allowed robust CAR T cell expansion and effector differentiation, resulting in enhanced antitumor efficacy and functional T cell persistence for 200 days. Regional T cell administration also promoted efficient elimination of extrathoracic tumor sites. This therapeutic efficacy was dependent on early CD4(+) T cell activation associated with a higher intratumoral CD4/CD8 cell ratios and CD28-dependent CD4(+) T cell-mediated cytotoxicity. In contrast, intravenously delivered CAR T cells, even when accumulated at equivalent numbers in the pleural tumor, did not achieve comparable activation, tumor eradication, or persistence. The ability of intrapleurally administered T cells to circulate and persist supports the concept of delivering optimal CAR T cell therapy through "regional distribution centers." On the basis of these results, we are opening a phase 1 clinical trial to evaluate the safety of intrapleural administration of mesothelin-targeted CAR T cells in patients with primary or secondary pleural malignancies.
| S-EPMC4373413 | biostudies-literature
Project description:To understand the molecular and neural mechanisms underlying alcohol addiction, many models ranging from vertebrates to invertebrates have been developed. In Drosophila melanogaster, behavioral paradigms from assaying acute responses to alcohol and to behaviors more closely modeling addiction have emerged in recent years. However, both the CAFÉ assay, similar to a two-bottle choice consumption assay, as well as conditioned odor preference, where ethanol is used as the reinforcer, are labor intensive and have low throughput. To address this limitation, we have established a novel ethanol consumption preference assay, called FRAPPÉ, which allows for fast, high throughput measurement of consumption in individual flies, using a fluorescence plate reader. We show that naïve flies do not prefer to consume ethanol, but various pre-exposures, such as ethanol vapor or voluntary ethanol consumption, induce ethanol preference. This ethanol-primed preference is long lasting and is not driven by calories contained in ethanol during the consumption choice. Our novel experience-dependent model of ethanol preference in Drosophila-a highly genetically tractable organism-therefore recapitulates salient features of human alcohol abuse and will facilitate the molecular understanding of the development of alcohol preference.
| S-EPMC3991769 | biostudies-literature