We exploited C MYC to

We exploited C-MYC to activate the endogenous c-Myc gene and enhance gene expression in neurosensory cell types. By doing so, we derived a self-renewing immortalized multipotent otic progenitor (iMOP) line from SOX2-expressing neurosensory precursors of the inner ear. We show that the endogenous C-MYC binds to most of the same promoters as SOX2 and amplifies transcripts that promote cell-cycle progression. This enhanced expression contributes to self-renewal but allows iMOP W 54011 to retain their capacity to differentiate into hair cells, supporting cells and neurons.

Results

Discussion

Experimental Procedures
For details regarding the materials and methods used in this work, see the Supplemental Experimental Procedures. All animal work conducted was approved by the IACUC at Harvard Medical School.

Author Contributions

Acknowledgments

Introduction
Primordial germ cells (PGCs) are the stem cells of the gametes, providing genome transmission to future generations (Lesch and Page, 2012). During development, PGCs undergo specification, migration, and proliferation. Reciprocal interactions between germ cells and somatic cells are important for gonadal differentiation (Kocer et al., 2009). However, little is known about the regulatory role of germ cells during sexual development.
In mammals, agametic male gonads develop into a normal testis cord, while loss of germ cells in ovaries at birth disrupts ovarian structures and folliculogenesis (Merchant-Larios and Centeno, 1981). In teleosts, the requirement of germ cells for gonadal development appears to be variable. Their absence leads to exclusive male development in medaka and zebrafish (Kurokawa et al., 2007; Siegfried and Nüsslein-Volhard, 2008; Slanchev et al., 2005), but not in goldfish or loach (Fujimoto et al., 2010; Goto et al., 2012).
Mammalian sex determination is regulated by antagonistic pathways, which direct the bipotential embryonic gonad toward ovarian or testicular fate (Warr and Greenfield, 2012). Moreover, evidence indicates that somatic sex needs to be reinforced throughout adulthood. In mice, loss of FOXL2 in mature ovary or DMRT1 in mature testis causes transdifferentiation of somatic cells (Matson et al., 2011; Uhlenhaut et al., 2009). In zebrafish, oocytes appear essential for the development of females in juveniles and for maintenance of the sexual phenotype in adults (Dranow et al., 2013).
The number of PGCs likely plays an important role in teleost sexual differentiation. For medaka and stickleback, females possess more germ cells than males due to their sexually dimorphic proliferation (Lewis et al., 2008; Saito et al., 2007). Transplantation of a single PGC into a germline-deficient zebrafish embryo generates males exclusively (Saito et al., 2008). ziwi mutants with reduced PGC numbers can develop as males or females; however, a greater reduction due to a hypomorphic allele in trans to a null allele gives rise to males (Houwing et al., 2007). These data argue that the absolute number of germ cells is important in determining the sexual phenotype of zebrafish.
Zebrafish are undifferentiated gonochorists since all individuals first initiate oogenesis via forming an immature ovary (Takahashi, 1974). In developing males, but not in females, a gonad transformation arises from apoptosis-driven degeneration of oocytes (Uchida et al., 2002; Wang and Orban, 2007) about 23–35 days postfertilization (dpf) leading to subsequent testis development (Orban et al., 2009; Uchida et al., 2002). Molecular control of sex determination and gonad differentiation in zebrafish appears to be complex (Liew and Orban, 2014; Orban et al., 2009) and variable across domesticated strains versus wild populations (Liew et al., 2012; Wilson et al., 2014).

Results

Discussion
In this report, we show that a dimorphic increase of germ cells occurs in the early larvae of zebrafish, similar to Japanese medaka (Satoh and Egami, 1972). Our data show that there is little change in the germ cell number during the first week of development. However, two distinct germ cell populations appear between 7 and 14 dpf, and this is strongly correlated with the resulting sex ratios of progeny. Thus, we propose that a sex-specific proliferation of germ cells marks the beginning of gonadal differentiation in zebrafish and individuals with a high PGC number have an increased propensity for the female fate.

Note that our EpiSC lines required continuous treatment with

Note that our EpiSC lines required continuous treatment with IWP-2 because removal of this Wnt inhibitor caused spontaneous differentiation. Since the original EpiSCs can be propagated without IWP-2, our EpiSCs must be distinguished from them by some intrinsic difference. There may be heterogeneities in Wnt signaling activity among epiblast cell populations, as previously observed for hESCs (Blauwkamp et al., 2012). Given that the inhibition of WNT secretion (or inhibition of Wnt signaling) promotes rapid EpiSC expansion, pyrilamine maleate with lower Wnt activity or responsiveness can be selected and established preferentially as EpiSCs under the original derivation conditions. On the other hand, IWP-2 blocks WNT secretion, leading to a reduced level of Wnt signaling. Since the WNT ligands available in culture would be greatly decreased, almost all of the epiblast cells, including cells with high Wnt responsiveness, would expand as EpiSCs. Therefore, EpiSCs derived by the IWP-2 method retain Wnt responsiveness and can differentiate in response to the Wnt stimulus. The fact that the 129C1 EpiSC line established by the original protocol (Brons et al., 2007) showed reduced responsiveness to WNT3A supports the notion described above.
Global gene-expression analysis revealed that addition of IWP-2 to 129C1 EpiSC cultures suppressed the expression of a specific cluster of genes. This tight cluster of genes was highly expressed in EpiSC lines obtained by the original protocol (i.e., 129C1 and Wt1 cells), whereas it was barely detectable in the EpiSCs derived by the IWP-2 method, in 129C1 cells cultured with IWP-2, or in epiblast tissues from E5.5–E7.5 embryos. This cluster of genes is involved in the regulation of development into the mesoderm, endoderm, or primitive streak. Interestingly, some of these genes (e.g., Cer1, Dkk1, and Sox17) have been considered to be characteristic markers of EpiSCs (Brons et al., 2007; Tesar et al., 2007; Kojima et al., 2014). However, we show here that the expression of these marker genes is not essential for the derivation and self-renewal of EpiSC lines. Our EpiSCs made by the IWP-2 method appear to represent authentic pluripotent EpiSCs, but with low levels of Wnt-induced spontaneous differentiation. In a study using XAV939 together with the Rho kinase inhibitor Y27632, Sumi et al. (2013) suggested that Wnt signaling inhibition plays a role in promoting less spontaneous differentiation. In the present study, we further demonstrated that genes suppressed by Wnt inhibition could be classified into at least two groups: one expressed in a partially differentiated subpopulation in EpiSC cultures (e.g., Gata4 or Sox17), and one expressed in SSEA1high undifferentiated EpiSCs, including Foxa2, Gsc, and Evx1. Expression of genes in both classes was dependent on Wnt signaling. Therefore, the Wnt inhibitor suppressed spontaneous, Wnt-induced differentiation to endoderm or mesoderm lineages, and also suppressed the expression of Wnt-dependent genes in SSEA1high pluripotent cells.

Experimental Procedures

Author Contributions

Acknowledgments

br Experimental Procedures For detailed

Experimental Procedures
For detailed experimental procedures, see the Supplemental Experimental Procedures.

Author Contributions

Acknowledgments

Introduction
Cell transplantation in congo red the central nervous system (CNS) requires exogenous cells to survive and integrate into the neural circuitry, thereby restoring function. The three major barriers to successful cell transplantation in adult tissue are distribution, survival, and integration of donor cells. The co-dependency of cell survival and cell integration on transplantation efficacy has been described (Ma et al., 2011).
Targets for cell therapy in the CNS, including retina and brain, have tissue-specific challenges that must be overcome for successful treatment. In conditions such as age-related macular degeneration and retinitis pigmentosa, transplanted outer retinal cells may be able to use the remaining inner retinal circuitry, and thus photoreceptor replacement is a feasible strategy to promote functional repair of the retina (Klassen et al., 2004). Although functional restoration after subretinal cell transplantation of neonatal or embryonic stem cell (ES)-derived post-mitotic rods into adult hosts has been demonstrated (Pearson et al., 2012; Lamba et al., 2009), the majority of studies have reported relatively low survival, from 0.04% to 8% on average. Similarly, in the brain, transplanted stem cells typically show low survival of 2%–8% (Nakagomi et al., 2009). Biomaterial approaches show promise in improving the efficiency of cell transplantation.
The hyaluronan (HA) and methylcellulose (MC) (HAMC) hydrogel is injectable, minimally swelling, bioresorbable, and fast gelling (Gupta et al., 2006; Baumann et al., 2010). It was shown to be superior to a number of different natural polymers in terms of physical and biological properties, including support of stem cell progeny survival and proliferation (Mothe et al., 2013; Ballios et al., 2010). The fast-gelling properties of HAMC are key to the more uniform distribution of cells in the retina and congo red compared to conventional saline delivery techniques.
The intimate relationship between cell survival and integration is investigated here with transplants of retinal stem cell (RSC)-derived rod photoreceptors. The development and characterization of adult RSC-derived rods in vitro (Ballios et al., 2012) closely resemble newborn post-mitotic rod photoreceptors in vivo (Akimoto et al., 2006), with expression of first immature (Nrl+ [Neural retina leucine zipper+]) and then mature (Rhodopsin+) rod markers in RSC progeny treated with taurine and retinoic acid (taurine/RA). Twelve-day in vitro differentiated rods (“immature” RSC-derived rods) express high levels of Nrl and low levels of Rhodopsin, whereas 28-day in vitro differentiated rods (“mature” RSC-derived rods) express high levels of both Nrl and Rhodopsin. Importantly, RSC-derived rods display electrophysiologic and functional light responsiveness in vitro that is similar to immature rod photoreceptors (Demontis et al., 2012). Transplantation of RSC-derived photoreceptors has demonstrated functional repair in early post-natal mouse models of disease (Inoue et al., 2010).
The role of HAMC in cell survival, integration, and, ultimately, functional repair was investigated in the retina with RSC-derived rods and in the brain with neural stem and progenitor cells (NSCs). In both tissues, cells delivered in HAMC survived significantly better than those delivered in conventional buffered saline vehicles. This survival effect was mediated by cell-material interactions through CD44, the putative HA receptor, and confirmed in vivo when transplanted CD44−/− RSC-derived rods no longer responded to the pro-survival effect previously observed with HAMC. In the retina, disruption of the outer limiting membrane (OLM) with dl-α-aminoadipic acid (AAA) (West et al., 2008) enhanced migration/integration of cells into the host outer nuclear layer (ONL). When delivered in HAMC, these integrated cells adopted mature rod morphology, expressed mature rod markers, and improved visual function in genetically blind mice. Unexpectedly, optimization of the delivery vehicle and host environment is sufficient to promote integration of mature rods, a population of cells previously considered unsuitable for transplantation (Pearson et al., 2012; MacLaren et al., 2006).

br Results br Discussion Drug

Results

Discussion
Drug therapy for the H/I-injured buy UNC2025 has focused largely on anti-oxidants (allopurinol, N-acetylcysteine), anti-excitotoxic agents (topiramate, memantine), and anti-inflammatory agents (cromolyn) (Juul and Ferriero, 2014; Hagberg et al., 2015). These therapies have been shown to primarily mediate their effects through neuroprotection and have resulted in varying degrees of functional recovery and a reduction in infarct volume. Further, while they display similarities in cost-effectiveness, implications in different injury models, and success in adults or old age animals, metformin is the only drug treatment that has demonstrated efficacy in generating new cells in the neonatal brain. Metformin may confer additional advantages such as improving lifespan (Martin-Montalvo et al., 2013) and promoting angiogenesis (Jin et al., 2014; Liu et al., 2014; Venna et al., 2014).
The finding that metformin, an anti-diabetic drug, increases the absolute number of NPCs following injury (rather than changing the relative percentage of differentiated cells) was surprising. Several possibilities could account for this observation. For example, metformin could have direct effects on NPCs (such as changes in cell-cycle kinetics and/or promoting cell survival) or indirect effects on non-NPCs that leads to the release of growth/trophic factors, providing immunomodulation, neuroprotection, and/or angiogenesis, which could effectively lead to an expansion in the size of the NPC pool. Indeed with respect to the latter, recent studies using chronic metformin treatment in adult ischemia models have demonstrated enhanced angiogenesis/neurogenesis correlating with motor function recovery (Jin et al., 2014; Liu et al., 2014). Given that the developing brain is known to exhibit a greater degree of plasticity (Kolb and Gibb, 2011) and a greater number of resident NPCs compared to the adult brain (Sachewsky et al., 2014), it is plausible that similar mechanisms of enhanced angiogenesis and neurogenesis are occurring in our H/I injury model.

Experimental Procedures

Author Contributions

Acknowledgments

Introduction
Multiple system atrophy (MSA) and Parkinson’s disease (PD) are adult-onset progressive neurodegenerative diseases that are hallmarked at the cellular level by the presence of alpha-synuclein (ΑSYN) protein containing inclusions. Interestingly, the inclusions are found in neurons in PD, where the SNCA gene encoding for ASYN protein is expressed, whereas they are prominent in oligodendrocytes as glial cytoplasmic inclusions (GCIs) in MSA (Papp et al., 1989; Stefanova et al., 2009). Because strong evidence showing that oligodendrocytes in the adult brain are capable of expressing SNCA is lacking, recent studies inspired from parkinsonian experimental models have proposed the interesting hypothesis that ASYN present in GCIs in MSA could be of neuronal origin and transfer to oligodendrocytes (Kisos et al., 2012; Reyes et al., 2014), where it would accumulate and potentially lead to oligodendrocyte dysfunction. However, more than a decade ago, Richter-Landsberg and coworkers reported on the transient expression of Snca in cultures enriched in oligodendrocytes, prepared by mechanical shaking of mixed rat glial primary brain cultures (Richter-Landsberg et al., 2000). This finding, however, was neither confirmed nor further explored, and in vivo evidences of ASYN expression during oligodendrocyte maturation are still missing. Consequently, follow-up studies mainly focused on understanding the functional consequences of wild-type or mutant human ASYN targeted expression in oligodendrocytes in experimental models in vivo and in vitro (Kragh et al., 2013; Yazawa et al., 2005).
To date, the origin of ASYN in GCIs in oligodendrocytes in MSA is still elusive, as is that of the few inclusions found in glial cells in the substantia nigra of people with PD (Wakabayashi et al., 2000). For these reasons, ASYN expression in oligodendrocytes remains to be firmly established, especially since investigations using patient material are still controversial (Asi et al., 2014; Miller et al., 2005). One way to explore ASYN expression in human oligodendrocytes is through the generation of human cellular models such as induced pluripotent stem cells (iPSCs), since patient oligodendrocytes are not always accessible postmortem.

The different stages of development of the two ICMs might

The different stages of development of the two ICMs might result from an uneven split of an initial single ICM during SCR 7 manufacturer collapse. A delay in formation of intercellular adhesions within the ICM might make the ICM prone to splitting (Togashi et al., 2015). The smaller part, in this case ICM1, might be lagging behind due to a lack of critical cell mass. However, we cannot exclude the possibility that ICM1 and ICM2 formed independently de novo at different time points (Noli et al., 2015). TE cells from the human non-expanded blastocysts are not fully committed; isolated and reaggregated TE cells are able to develop into blastocysts with ICM cells expressing NANOG (De Paepe et al., 2013). Either way, our case report suggests new avenues to studies of cellular and molecular events behind MZ twinning and discordant growth, commonly associated with monochorionic/diamniotic pregnancies, as well as lineage commitment at the earliest stages of human development.

Experimental Procedures

Author Contributions

Acknowledgments

Introduction
Proper development of many tissues and organs (e.g., gut, kidney, blood vessels, lung, etc.) requires the formation of lumenal structures of various shapes (Shao et al., 2015). Indeed, one of the first behaviors of early embryonic epiblast cells is formation of the lumen of the proamniotic cavity SCR 7 manufacturer (Luckett, 1975; Rossant and Tam, 2009). This process is still poorly understood but is essential for the further successful development of the embryo. In vitro, many stem cells grow into organoids with lumenal structures (Lancaster and Knoblich, 2014), indicating that self-organization to form lumens is intrinsic to a variety of stem cell types. Because proper morphogenesis and function are so dependent on lumenal integrity in many settings, a detailed understanding of the lumen-forming process and the mechanisms underlying it is critical for the proper engineering of transplantable tissues.
Much of what we currently know about lumen formation comes from the study of transformed, tissue-specific cell lines such as Madin-Darby canine kidney type 2 (MDCK.2) and Caco-2 (human colorectal cancer) cells; these cells form polarized lumenal cysts de novo when embedded in extracellular matrix (ECM) complex (Martin-Belmonte and Mostov, 2008; Rodriguez-Boulan and Macara, 2014). Using these models, it has been demonstrated that lumen formation is initiated during the first cell division by the trafficking of apical proteins such as Ezrin, Podocalyxin, and Crumbs3 from the cell periphery to the nascent cytokinetic plane (Bryant et al., 2014; Schlüter et al., 2009). This process allows the establishment of the apical membrane initiation site (AMIS), an actin-rich region that matures to become the lumen (Martin-Belmonte and Mostov, 2008; Rodriguez-Boulan and Macara, 2014). Although MDCK.2 and Caco-2 are useful to model lumen formation in differentiated models (kidney and gut), effective general tools to model development of early embryonic tissues that undergo de novo lumen formation are currently lacking.
We have discovered that when dissociated human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) are plated at low density in 2D or 3D conditions, the first mitotic event frequently generates a two-cell cyst with an AMIS-like domain that matures to a lumen. The lumen-forming capacity of pluripotent stem cells (PSCs) is amenable to manipulation to generate lumens of complex shapes using micro-engineered substrates. Molecularly, we find that, as in MDCK.2 cells, augmenting ROCK (Rho-associated kinase)-MYOSIN-II signaling, which leads to the formation of actin stress fibers (Burridge and Wennerberg, 2004), inhibits apical lumen formation in PSC (Rodríguez-Fraticelli and Martín-Belmonte, 2013). Additionally, we demonstrate a critical role for two separate actin polymerization processes (via mammalian diaphanous-related formin 1 [MDIA] and via ARP2/3) in lumenogenesis. Overall, our data establish PSCs as effective non-transformed and undifferentiated cells to be identified as a robust model for lumenogenesis.

In addition we observed significantly

In addition, we observed significantly elevated levels of synapsin and dopamine in Parkin cultures, although the changes in PINK1 cells did not reach statistical significance when compared with control iPSC- or ESC-derived floor-plate mDA neurons. Increased DA levels may appear, in contrast to the well-known loss of DA in the AZD-9291 of PD patients. While reduction in the PD brain may simply reflect the progressive loss of DA-producing neurons, there are data suggesting a reduction in DA levels or release in the remaining cells (Nguyen et al., 2011). Our HPLC data argue for an increase in intracellular dopamine levels in PD AZD-9291 versus control iPSC-derived mDA neurons, which may be indicative of dysregulation of neurotransmitter release linked to changes in synaptic activity. In support of our findings, previous animal studies have also reported increased levels of DA and DOPAC in Parkin- and PINK1-deficient mice (Kitada et al., 1998). Alternatively, this could be due to a loss of ATP and the proton gradient, which determines the synaptic vesicle-to-cytosol equilibrium of DA. It will be important to address whether such changes are limited to the Parkin, or potentially, the PINK1 phenotype or reflect changes in other genetic or sporadic forms of PD at distinct stages of disease. It is tempting to speculate whether increased levels could contribute to the pathogenic cycle induced by mitochondrial dysfunction and ultimately lead to mDA neuronal cell death (Figure 6).
Determining the role of α-synuclein in PD pathogenesis is complicated by the fact that the physiological role of α-synuclein is not fully known; nevertheless, studies have linked α-synuclein to a variety of functions including the regulation of synaptic membrane processes and regulation of neurotransmitter release (Davidson et al., 1998; Nemani et al., 2010). Notably, α-synuclein has also been found to act as a negative regulator of synaptic vesicle fusion and exocytotic DA release, and studies have found that its overexpression impairs DA transmission in the early phases of neurodegeneration (Gaugler et al., 2012; Platt et al., 2012). Along these lines, other groups have found that absent or mutated α-synuclein can affect the compartmentalization of presynaptic DA and alter DA storage-pool capacity (Abeliovich et al., 2000; Mosharov et al., 2006; Murphy et al., 2000; Yavich et al., 2004). Furthermore, phage display and nuclear magnetic resonance spectroscopy have revealed that synapsin Ia is a binding partner of α-synuclein, which suggests that their interaction may play a critical role in the release of neurotransmitters (Woods et al., 2007). These results indicate that increased intracellular levels of DA observed in PD iPSC mDA neurons could be related to vesicular trafficking defects caused by α-synuclein protein dysfunction and other synaptic changes. Increased reactive oxygen species (ROS) resulting from defective mitochondria in PD mDA neurons may trigger destruction of synaptic vesicles, disrupt proper encapsulation of DA within vesicles, and cause the loss of the proton gradient required for synaptic vesicles to accumulate DA against its concentration gradient. Such abnormal DA homeostasis could eventually perpetuate a cascade of oxidative stress within already vulnerable cells.

Experimental Procedures

Author Contributions

Introduction
Human induced pluripotent stem cells (hiPSCs) are remarkable for their ability to self-renew and differentiate into various tissues and cell types (Takahashi and Yamanaka, 2006). Differentiation of these cells into a specific cell type allows for in-depth study with the goal of cell replacement therapy for neurodegenerative diseases such as Parkinson\’s disease (PD), which is caused by selective loss of dopaminergic (DA) neurons in the substantia nigra pars compacta in the midbrain (Xu et al., 2013b). Expanding our knowledge of the cellular and molecular factors involved in the maturation and differentiation of human-derived neurons will significantly boost our ability to manipulate and generate specific cell types for disease therapy.

We have developed an in vivo model in which

We have developed an in vivo model in which BALB/c mice synthesize DNRAbs following immunization with a configuration of the consensus sequence multimerized on a polylysine backbone (termed MAP-DWEYS), while BALB/c mice immunized with the polylysine backbone alone (MAP-core) do not (Kowal et al., 2004). This model allows us to evaluate DNRAbs as causal agents of neuronal injury, independent of other autoantibodies and the high levels of systemic inflammatory mediators found in spontaneous mouse SLE models (Sakic, 2012). Circulating DNRAbs cause no detectable ABT199 pathology in MAP-DWEYS immunized mice with an intact BBB. However, upon exposure to LPS, mice have 20–25% loss of hippocampal neurons (occurring within the first week post-LPS) and persistent memory impairment, assessed in the T-maze and the Morris water maze (Kowal et al., 2004).

Materials and Methods

Results

Discussion
This study shows that SLE patients carrying circulating DNRAbs display a selective impairment in spatial cognition. While SLE patients commonly complain of problems with spatial navigation, such as not knowing whether they are moving toward or away from home, or not knowing if they are on their block or a block away, this study is unique in that spatial performance has been explicitly tested and has been related to serology. Previous studies have demonstrated that DNRAbs can be found in the CSF of SLE patients at concentrations capable of altering the strength of murine NMDAR-mediated synaptic potentials and causing excitotoxic neuronal death in mice in vivo, or death of human NMDAR-expressing cell lines (DeGiorgio et al., 2001; Faust et al., 2010; Fragoso-Loyo et al., 2008). Moreover, DNRAbs in the CSF have been strongly associated with non-focal CNS disease (Arinuma et al., 2008; Fragoso-Loyo et al., 2008; Gono et al., 2011; Yoshio et al., 2006), while neuroimaging and neuropathologic studies of SLE patients have identified the hippocampus as a region of frequent abnormality (Appenzeller et al., 2006; Ballok et al., 2004).
This study shows that mice in which DNRAbs penetrate into the hippocampus display a clear disruption in the CA1 place cell system, which is a key part of the neural substrate for spatial navigation (O\’Keefe, 2007). Also, some crucial properties of place cells are NMDAR-dependent (Ekstrom et al., 2001; Kentros et al., 1998). Importantly, the significant DNRAb-mediated expansion in place field size of CA1 neurons likely leads to a spatial map with lower resolution. Similar alterations in place cell firing have been described in a mouse model of Alzheimer\’s disease (Cacucci et al., 2008), as well as mice with a hippocampal-specific deletion of the gene encoding GluN1 (McHugh et al., 1996). Since NMDARs are located in dendritic spines, we also analyzed CA1 dendrites and confirmed reduced number of dendritic branches and dendritic spines in CA1 neurons. A similar abnormality was seen in the CA1-projecting CA3 neurons that may reflect direct exposure to DNRAbs or alternatively, retrograde damage to the Schaffer commissural collateral CA3 axons that project to CA1 neurons (Wang et al., 2012). Surprisingly, both functional and structural neuronal damage evolved after the inciting trigger was no longer present, and persisted for at least 2months after the BBB breach. This slow evolution of dysfunction is consistent with the clinical data on NPSLE. Changes in cognitive function are insidious and most commonly do not occur concurrent with flares in disease activity or with overt evidence of CNS inflammation (Shimojima et al., 2005).
SLE-prone mice, such as the NZB/W and MRL/lpr strains, have been previously studied and show a marked impairment in spatial memory that develops once both autoantibodies and inflammatory cytokines are elevated (Sakic, 2012). Thus, the contribution of a specific antibody subset cannot be assessed in these mice. We found that mice with hippocampal exposure to DNRAbs were not impaired in the NOR task. By contrast, hippocampal exposure to DNRAbs correlates with spatial memory impairment in four distinct assessments: the T-maze, the Morris water maze, the clock maze, reported previously (Kowal et al., 2004, 2006), and the OPM task described here. Since SLE patients carrying elevated titers of DNRAbs exhibited impaired spatial memory compared to HC subjects, the mouse model appears to be a valid model for NPSLE. Changes in dendritic complexity in hippocampal neurons have been reported in a model of anti-phospholipid syndrome, although the mechanism for neuronal destruction in unknown (Frauenknecht et al., 2014). There are numerous brain-reactive antibodies in SLE (Yaniv et al., 2015). It will be important to determine how each might contribute to manifestations of NPSLE.

br Conclusions The following are the supplementary data related to

Conclusions
The following are the supplementary data related to this article.

Author Contributions

Conflict of Interest

Acknowledgements
This work was supported by the Health Research Institute of University Hospital Clínico San Carlos, “Generalitat Valenciana” (PROMETEOII/2014/065), by the Spanish Ministry of Economy and Competitiveness (SAF2012-31187, SAF2013-49788-EXP, SAF2015-65878 from MINECO), Instituto de Salud Carlos III (PIE14-00045 and Redes Temáticas de Investigación en SIDA RETICRD12/0017/0029 and RD12/0017/0037), and the Junta de Andalucía (Proyecto de Excelencia CTS-6313). The present investigation was also funded by the Spanish Ministry of Economy and Competitiveness and the Federal Ministry of Education and Research (BMBF) within the ERA NET PathoGenoMics2 program, grant number 0315441A. This work was further funded by grants BIO2011-25012 and BIO2014-54494-R from the Spanish Ministry of Economy and Competitiveness. The authors gratefully acknowledge the financial support provided by the European Regional Development Fund (ERDF). C.B and D.R would like to acknowledge funding from the Spanish Ministry of Economy and Competitiveness (CTQ2014-55279-R). J.F.V.C. was supported by a fellowship “Ayudas Predoctorales de Formación en Investigación en Salud” from the Instituto de Salud Carlos III (Spain) and the CONACYT-SECITI (México). S.S.V. is supported by a grant from the Spanish Ministry of Science and Innovation (Contratos Juan Rodés, ECC/1051/2013), and T.S. is supported by a grant from the European Society of Pediatric Infectious Diseases (ESPID). Funding Agencies did not have any role neither in the writing of the manuscript or the decision to submit it Cisplatin nor have paid to write this article. The authors wish to acknowledge the participation of all of the study participants who contributed to this work as well as the clinical research staff of the participating institutions who made this research possible.

Introduction
Latently infected CD4+ T cells are the major barrier to HIV-1 cure efforts. The cells contain integrated proviruses that are transcriptionally silent and thus able to evade detection and clearance by the immune system. The shock-and-kill cure strategy seeks to first reactivate these latent viruses without causing global T cell activation followed by clearance of the reactivated cells by the immune system (reviewed in Siliciano and Siliciano, 2013; Archin and Margolis, 2014). Latency reactivating agents (LRAs) are drugs that induce HIV-1 transcription. Notable drug classes include PKC agonists and HDAC inhibitors (HDACi), which have been very effective in inducing HIV-1 transcription in cell lines (Contreras et al., 2009; Xing et al., 2011; Li et al., 2013; DeChristopher et al., 2012). Unfortunately, in vitro experiments with primary resting CD4 T cells from patients on suppressive antiretroviral therapy (ART) regimens suggest that most individual LRAs are unable to induce substantive amounts of HIV-1 transcription with the notable exception of PKC agonists bryostatin-1-1 (Bullen et al., 2014) and ingenol (Spivak et al., 2015). However, LRA combinations in the same system are capable of inducing significant HIV-1 transcription (Laird et al., 2015; Jiang et al., 2015; Darcis et al., 2015).
The other half of the cure strategy deals with killing newly reactivated infected CD4+ T cells. Recent experiments suggest that reactivation from latency is not enough to induce cell death (Shan et al., 2012), and therefore there may be a need for immune mediated eradication. Expanded CD8+ T cell lines were able to clear reactivated latently infected resting CD4+ T cells following exposure to the HDAC inhibitor, vorinostat (Sung et al., 2015). However primary CD8+ T cells from patients on suppressive ART regimens that were pre-stimulated with overlapping Gag peptides were unable to consistently reduce the amount of HIV-1 mRNA induced from autologous resting CD4+ T cells that were activated with PMA and ionomycin (Walker-Sperling et al., 2015).

atipamezole br Methods br Results br Discussion The

Methods

Results

Discussion
The intriguing relationship between commensal microbes and atherosclerosis has received increasing attention over the past few years. However, the specific mechanisms whereby commensal microbes regulate the development of atherosclerosis are just beginning to be elucidated (Brown and Hazen, 2015; Koren et al., 2011; Serino et al., 2014; Tang and Hazen, 2014). Recently, Spence et al. found that a number of metabolites from proteins/amino acids in the diet, including p-cresyl sulfate, indoxyl sulfate, and others (Spence et al., 2016), might contribute to development of cardiovascular disease (CVD). Previous studies have identified the pathway linking dietary lipid intake, intestinal microflora, and atherosclerosis. These studies indicated that increasing the dietary intake of precursors of toxic metabolic products of the intestinal microbiome, such as the trimethylamine-N-oxide from phosphatidylcholine and other forms of choline and carnitine (Koeth et al., 2013; Tang et al., 2013; Wang et al., 2011), was associated with CVD. The pathway of the metabolism of these toxic metabolic products of the intestinal microbiome represents a unique additional nutritional contribution to the pathogenesis of CVD. In fact, numerous studies have shown that commensal microbe-derived metabolites can act as hormones modulating CVD risk (Brown and Hazen, 2015). Metabolism-independent pathways, in particular the role of the immune system in commensal microbe-derived atherosclerosis, remain largely unexplored.
Our study showed that serum lipid levels were significantly increased in WD-fed mice regardless of AT or not, which is in line with previous observations (Fu et al., 2015; Le Chatelier et al., 2013; Velagapudi et al., 2010). However, it remains unclear how the gut microbiota affects serum lipid metabolism and systemic lipid metabolism in adipose tissue. It is thought that hyperlipidemia (especially LDL-C) generates an adaptive immune response mediated by autoantibody that is produced by activated atipamezole and then induces atherosclerosis (Hermansson et al., 2010; Hilgendorf et al., 2014). However, in the present study, there was no correlation between lipid levels and atherosclerosis development. Therefore, we determined whether B2-cell activation is mediated by microbiota rather than by hyperlipidemia in atherosclerosis. Here, we confirmed, by eliminating the intestinal microbiota and depleting B2 cells in the WD-fed mice, that hyperlipidemia did not directly potentiate atherosclerosis by altering B2-cell activation. Our results provide novel evidence that B2 cells are causally related to microbiota-induced atherosclerosis. Our findings also indicate that proatherogenic effects do not solely depend on hypercholesterolemia-induced immune response. Clinically, these findings may explain why only control of lipids is not effective preventive therapy in some patients with atherosclerosis.
B cells play a complex role in the development of atherosclerosis via antibody production. In particular, the role of B2 cells in atherosclerosis is highly debatable (Kyaw et al., 2010; Tsiantoulas et al., 2014). As such, understanding the impact of B2 cells on atherosclerosis and elucidating factors that regulate their activity are important. In the present study, our observed differences in FO B cells of PVAT between the WD group and WD-AT group suggest that FO B cells are missing from the PVAT in the WD-AT group, potentially due to commensal microbe deletion by broad-spectrum antibiotics. However, it remains controversial whether commensal microbes directly induce B2-cell activation during the development of atherosclerosis and whether activated B2 cells play a critical role in this process. Thus, future studies are needed to determine whether B2 cells are essential for commensal microbe-derived atherogenesis and to elucidate potential atherogenic pathways. It is known that one of the early consequences of B2 cell activation is the up-regulation of costimulatory molecules, such as MHC class II molecules, which can serve to enhance B cell interactions and present antigen (Jiang et al., 2013; Jin et al., 2014). Another major outcome of B2-cell activation is the production of large amounts of specific antibodies (Shapiro-Shelef and Calame, 2005). The results of our study apparently showed that a greater proportion of B2 cells expressed MHC class II molecules in the spleen of WD-fed mice. We also detected elevated levels of IgG. In particular, the IgG subclass IgG3 displayed the highest activity in serum, whereas AT significantly reduced the expression of MHC class II molecules and the levels of IgG and IgG3. These results support the concept that microbiota-induced atherosclerosis is associated with B2-cell activation.

This study found that the median CPT over time

This study found that the median CPT over time showed a greater decrease in the control group than the tranylcypromine group. While both groups were given ranibizumab injections at baseline and week 4, the gene therapy group patients received a core vitrectomy, associated with subretinal gene therapy, at day 7. This may have removed any remaining active ranibizumab from the first intravitreal injection, thus leading to a smaller initial response in the gene therapy group. In most studies (CATT Research Group et al., 2011; Lalwani et al., 2009), the greatest decreases in CPT occurred in the pectin first 2 months of the study.

Conflict of Interest

Role of Funding Sources
Funding Sources: National Health and Medical Research Council of Australia (AP1010405), Lions Eye Institute, Perth Australia, Avalanche Biotechnologies, Menlo Pk, CA, USA. Avalanche Biotechnologies had no role in the design but participated in the data management and data analysis of the manuscript. The other two funding agencies contribution was only financial.

Authors Contribution

Acknowledgements