Immature State Research Articles

Information Communications Technology and Economic Growth in Sub-Saharan Africa: A Panel Data Approach

This research empirically analyzed the impact of mobile phone and the Internet in SubSaharan Africa (SSA) for the period of 2006-2015 using unbalanced panel data of 40 countries. We have employed the robust two-step system GMM. Results showed that growth in mobile phone penetration has contributed significantly to the GDP per capita of the region after controlling for a number of other variables. A 10% increase in mobile phone penetration results in a 1.2% change in real GDP per capita. Therefore, improving access to mobile phones will play a critical role in reducing the poverty level of the region through raising the per capita income of the population. However, the Internet has not contributed to the per capita GDP during the study period. The insignificant impact of the Internet could be due to low penetration of the technology, low ICT skill of Internet users, lack of or insufficient local content on the global network, the relatively immature state of the technology in the region, and a possible misuse of it. Therefore, governments and other stakeholders should design policies that encourage expansion of the Internet until a critical mass of users is achieved. In addition to improving Internet access, policies which focus on ICT skill development and local content creation should also be designed and implemented. Donors and the international community should also give their support directly to those startups which focus on local content production and distribution.

Different Expression of Interferon-Stimulated Genes in Response to HIV-1 Infection in Dendritic Cells Based on Their Maturation State.

Dendritic cells (DCs) are professional antigen-presenting cells whose functions are dependent on their degree of differentiation. In their immature state, DCs capture pathogens and migrate to the lymph nodes. During this process, DCs become resident mature cells specialized in antigen presentation. DCs are characterized by a highly limiting environment for human immunodeficiency virus type 1 (HIV-1) replication due to the expression of restriction factors such as SAMHD1 and APOBEC3G. However, uninfected DCs capture and transfer viral particles to CD4 lymphocytes through a trans-enhancement mechanism in which chemokines are involved. We analyzed changes in gene expression with whole-genome microarrays when immature DCs (IDCs) or mature DCs (MDCs) were productively infected using Vpx-loaded HIV-1 particles. Whereas productive HIV infection of IDCs induced expression of interferon-stimulated genes (ISGs), such induction was not produced in MDCs, in which a sharp decrease in ISG- and CXCR3-binding chemokines was observed, lessening trans-infection of CD4 lymphocytes. Similar patterns of gene expression were found when DCs were infected with HIV-2 that naturally expresses Vpx. Differences were also observed under conditions of restrictive HIV-1 infection, in the absence of Vpx. ISG expression was not modified in IDCs, whereas an increase of ISG- and CXCR3-binding chemokines was observed in MDCs. Overall these results suggest that sensing and restriction of HIV-1 infection are different in IDCs and MDCs. We propose that restrictive infection results in increased virulence through different mechanisms. In IDCs avoidance of sensing and induction of ISGs, whereas in MDCs increased production of CXCR3-binding chemokines, would result in lymphocyte attraction and enhanced infection at the immune synapse.IMPORTANCE In this work we describe for the first time the activation of a different genetic program during HIV-1 infection depending on the state of maturation of DCs. This represents a breakthrough in the understanding of the restriction to HIV-1 infection of DCs. The results show that infection of DCs by HIV-1 reprograms their gene expression pattern. In immature cells, productive HIV-1 infection activates interferon-related genes involved in the control of viral replication, thus inducing an antiviral state in surrounding cells. Paradoxically, restriction of HIV-1 by SAMHD1 would result in lack of sensing and IFN activation, thus favoring initial HIV-1 escape from the innate immune response. In mature DCs, restrictive infection results in HIV-1 sensing and induction of ISGs, in particular CXCR3-binding chemokines, which could favor the transmission of HIV to lymphocytes. Our data support the hypothesis that genetic DC reprograming by HIV-1 infection favors viral escape and dissemination, thus increasing HIV-1 virulence.

Effect of Matrix Metallopeptidase 13 on the Function of Mouse Bone Marrow-derived Dendritic Cells.

Background:Dendritic cells are professional antigen-presenting cells found in an immature state in epithelia and interstitial space, where they capture antigens such as pathogens or damaged tissue. Matrix metallopeptidase 13 (MMP-13), a member of the collagenase subfamily, is involved in many different cellular processes and is expressed in murine bone marrow-derived dendritic cells (DCs). The function of MMP-13 in DCs is not well understood. Here, we investigated the effect of MMP-13 on DC maturation, apoptosis, and phagocytosis.Methods:Bone marrow-derived dendritic cells were obtained from C57BL/6 mice. One short-interfering RNA specific for MMP-13 was used to transfect DCs. MMP-13-silenced DCs and control DCs were prepared, and apoptosis was measured using real-time polymerase chain reaction and Western blotting. MMP-13-silenced DCs and control DCs were analyzed for surface expression of CD80 and CD86 and phagocytosis capability using flow cytometry.Results:Compared to the control DCs, MMP-13-silenced DCs increased expression of anti-apoptosis-related genes, BAG1 (control group vs. MMP-13-silenced group: 4.08 ± 0.60 vs. 6.11 ± 0.87, P = 0.008), BCL-2 (control group vs. MMP-13-silenced group: 7.54 ± 0.76 vs. 9.54 ± 1.29, P = 0.036), and TP73 (control group vs. MMP-13-silenced group: 4.33 ± 0.29 vs. 5.60 ± 0.32, P = 0.001) and decreased apoptosis-related genes, CASP1 (control group vs. MMP-13-silenced group: 3.79 ± 0.67 vs. 2.54 ± 0.39, P = 0.019), LTBR (control group vs. MMP-13-silenced group: 9.23 ± 1.25 vs. 6.24 ± 1.15, P = 0.012), and CASP4 (control group vs. MMP-13-silenced group: 2.07 ± 0.56 vs. 0.35 ± 0.35, P = 0.002). Protein levels confirmed the same expression pattern. MMP-13-silenced groups decreased expression of CD86 on DCs; however, there was no statistical difference in CD80 surface expression. Furthermore, MMP-13-silenced groups exhibited weaker phagocytosis capability.Conclusion:These results indicate that MMP-13 inhibition dampens DC maturation, apoptosis, and phagocytosis.

Hydrocarbon Potential Assessment for Dokan and Gulneri Formations (Upper Cretaceous) in K-130 Well Kirkuk Oil Field, Kurdistan, North of Iraq

Eight rock samples from the Dokan and Gulneri formations in one subsurface section of Kirkuk-130 Well in Northern Iraq were chosen to be studied optically and analytically to determine the source- rock properties of the both rock units. The studied samples showed richness in amorphous organic matter with generally low percentages for other organic matter components. Three palynofacies were distinguished representing proximal suboxic-anoxic shelf and shelf- to – basin transition for the Dokan Formation, whereas a distal suboxic-anoxic basin appeared to be the paleodepositional environment for the Gulneri Formation.The Dokan Formation appears to be poor to fair, whereas the Gulneri Formation showed very good to excellent richness in organic matter as measured from total organic carbon (TOC) contents. The Rock- Eval Pyrolysis data for the studied formations showed that the quality of organic matter in the Dokan Formation is mostly type II/III kerogen, whereas in the Gulneri Formation it is mostly type I kerogen. Thermal maturity indicators, as observed from pyrolysis parameters, indicate an immature state for both studied formations. Accordingly, it is concluded that the Dokan and Gulneri formations did not contribute to generating the accumulated oil in the Kirkuk Field.

The therapeutic effect and its mechanism of dendritic cells overexpressed suppressors of cytokine signaling 1 on acute liver failure in mice

Objective To investigate the impact of suppressor of cytokine signaling 1 (SOCS1) overexpression on dendritic cells (DC) functions and its therapeutic effect on acute liver failure (ALF) in mice. Methods Bone marrow derived dendritic cells (BMDC) from C57BL/6 mice were transfected with lentivirus encoding SOCS1 and negative control lentivirus at a MOI=50, and labeled as DC-SOCS1and DC-VNG, respectively after 96 hours of successful transduction. Then DCs were stimulated with lipopolysaccharides(LPS)1 mg/L and collected for flow cytometry analysis of surface costimulatory molecules, allogeneic mixed lymphocyte reaction (MLR) and western blot test of Janus kinase (JAK)/signaling transducers and activators of transcription (STAT) pathway. Afterwards, 90 mice were randomly assigned into 4 groups including 12 in normal control group, 26 in ALF group, 26 in treatment groups with DC-SOCS1 and 26 with the treatment of DC-VNG. All were received tail vein injection with normal saline, modified DC-VNG and DC-SOCS1 suspended in normal saline, respectively. Twelve hours after injection, LPS (10 μg/kg)/D-GaIN (600 mg/kg) were injected intraperitoneally to induce ALF model. The mortality, serum levels of alanine aminotransferase (ALT) and aspartate transaminase (AST), liver pathology and proportion of splenic regulatory T cells of each group were observed. Means in different groups were compared with one-way ANOVA analysis. Categorical variables were analyzed with χ2 test. Variables were examined with normality test and homogeneity of variance with LSD test. Results The results of mixed lymphocyte reaction (MLR) revealed that T cell proliferation ratio in DC-SOCS1 group with mixture ratio of 100∶1 were (25.87±0.38)%, which was lower than that of mixture ratio of 10∶1 in the mDC group ([84.29±3.25]%) with statistical significance (χ2=49.821, P<0.01); interleukin (IL)-10 concentration was higher than that in mDC group with mixture ratio of 10∶1 with statistical significance (F=20.112, P<0.05); IL-6 concentration was also lower with statistical significance (F=47.718, P<0.05). Compared to imDC, expression of JAK2 (t= 0.525, 0.523 and 0.489, respectively, all P<0.01), signal transduction factors and activation of transcription factors-1 (STAT1) (t=0.442, 0.400 and 0.402, respectively, all P<0.01) and SOCS1 (t=0.322, 0.363 and 1.090, respectively, all P<0.01) of mDC, DC-VNG and DC-SOCS1 after LPS stimulation increased significantly. Furthermore, the expressions of phosphorylated STAT1 (p-STAT1) and phosphorylated JAK2 (p- JAK2) of DC- SOCS1 were much lower than those of the mDC, with statistically significant difference (t=-3.840 and 0.254, respectively, both P<0.01). Pathological analysis revealed that there existed moderate hepatic cells necrosis and less immune cell infiltration in DC-SOCS1 group accompanied with higher regulatory T lymphocytes proportion than those in ALF group and DC-VNG group. Survival rate of ALF with DC-SOCS1 treatment group was significantly higher than that of ALF group with statistical difference (χ2=12.87, P<0.05). Conclusions DC-SOCS1could sustain an immature state and exhibit as regulatory DC through negative regulation of JAK2/STAT1 pathway with overexpression of SOCS1. Infusion of DC-SOCS1 could ameliorate ALF by inhibiting aggressive inflammation response with increased proportion of regulatory T cells in mice, which shows good therapeutic effect for ALF mice. Key words: Liver failure, acute; Suppressor of cell signaling 1; Dendritic cells

Dendritic cells with single Ig interleukin-IR-related molecule gene overexpression exhibit immature properties and prolong allograft survival

Objective To study the effect of single Ig interleukin (IL)-IR-related molecule (SIGIRR) on maturation of dendritic cells (DCs) and rejection of allografts in mice. Methods Murine bone marrow cells were extracted and induced into DCs. Adenoviral vector coding SIGIRR (Ad-SIGIRR) was constructed to increase SIGIRR gene expression in bone marrow-derived DCs. The phenotype and cytokine secretion of DCs transfected with Ad-SIGIRR (DC-SIGIRR) and control group in the absence and the presence of lipopolysaccharides (LPS) were analyzed. In vivo, C57BL/6 mice, which were pretreated with DC-SIGIRR or control treatment via intravenous injection, received islet transplantation. Intraperitoneal glucose tolerance test (IPGTT), graft survival time and histological changes were observed. Results DC-SIGIRR remained relative immature state after LPS stimulation. DC-SIGIRR expressed lower levels of costimulatory (CD40, CD80, CD86) and major histocompatibility complex (MHC) molecules, secreted lower levels of IL-12 [(547.80±84.67) pg/ml, P=0.026] and higher levels of IL-10 [(410.40±36.11) pg/ml, P=0.031] than in the control group. IPGTT suggested the blood glucose peak in SIGIRR group was statistically lower than in the other groups [(19.0±1.0) mmol/L, P=0.041]. Histological examination revealed less immune damage of the islet grafts, higher secretion activity and significantly longer allograft survival time [(14.3±3.6) days, P=0.036] in DC-SIGIRR pretreatment group. Conclusion SIGIRR inhibits DC maturation and DC transfected with SIGIRR prolongs allografts survival, which suggests therapeutic potential to prevent rejection in organ transplantation. Key words: Single Ig interleukin-IR-related molecule; Gene transfect; Dendritic cell; Islet transplantation; Rejection

Phenotypic and functional consequences of different isolation protocols on skin mononuclear phagocytes.

Mononuclear phagocytes are present in skin and mucosa and represent one of the first lines of defense against invading pathogens, which they detect via an array of pathogen-binding receptors expressed on their surface. However, their extraction from tissue is difficult, and the isolation technique used has functional consequences on the cells obtained. Here, we compare mononuclear phagocytes isolated from human skin using either enzymatic digestion or spontaneous migration. Cells isolated via enzymatic digestion are in an immature state, and all subsets are easily defined. However, cells isolated by spontaneous migration are in a mature state, and CD141 cross-presenting DCs (cDC1) are more difficult to define. Different pathogen-binding receptors are susceptible to cleavage by blends of collagenase, demonstrating that great care must be taken in choosing the correct enzyme blend to digest tissue if carrying out pathogen-interaction assays. Finally, we have optimized mononuclear phagocyte culture conditions to enhance their survival after liberation from the tissue.

Taking Stock: Estimating Vulnerability Rediscovery

How often do multiple, independent parties discover the same vulnerability? There are ample models of vulnerability discovery, but little academic work on this issue of rediscovery. The immature state of this research and subsequent debate is a problem for the policy community, where the government’s decision to disclose a given vulnerability hinges in part on that vulnerability’s likelihood of being rediscovered and used maliciously by another party. Research into the behavior of malicious software markets and the efficacy of bug bounty programs would similarly benefit from an accurate baseline estimate for how often vulnerabilities are discovered by multiple independent parties. This paper presents a new dataset of more than 2,600 vulnerabilities, and estimates vulnerability rediscovery across different vendors and software types. It concludes that rediscovery happens more often than the 1% to 9% range previously reported. The aggregate rediscovery rate for our dataset is 12.7%, ranging between 10.8% for Chrome between 2009 and 2017, to 21.9% for Android between 2016 and 2017. For Android and Chrome, more than 60% of all rediscovery takes place in the first month after the original vulnerability’s disclosure. These results are largely in line with those of the original version of this paper published in July 2017, and indicate that the information security community should map the impact of rediscovery on the efficacy of bug bounty programs, and policymakers should more rigorously evaluate the costs and requirements for non-disclosure of software vulnerabilities.

Impact of Cystinosin Glycosylation on Protein Stability by Differential Dynamic Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)

Cystinosis is a rare autosomal recessive lysosomal storage disorder characterized by intralysosomal accumulation of cystine. The causative gene for cystinosis is CTNS, which encodes the protein cystinosin, a lysosomal proton-driven cystine transporter. Over 100 mutations have been reported, leading to varying disease severity, often in correlation with residual cystinosin activity as a transporter and with maintenance of its protein-protein interactions. In this study, we focus on the ΔITILELP mutation, the only mutation reported that sometimes leads to severe forms, inconsistent with its residual transported activity. ΔITILELP is a deletion that eliminates a consensus site on N66, one of the protein's seven glycosylation sites. Our hypothesis was that the ΔITILELP mutant is less stable and undergoes faster degradation. Our dynamic stable isotope labeling by amino acids in cell culture (SILAC) study clearly showed that wild-type cystinosin is very stable, whereas ΔITILELP is degraded three times more rapidly. Additional lysosome inhibition experiments confirmed ΔITILELP instability and showed that the degradation was mainly lysosomal. We observed that in the lysosome, ΔITILELP is still capable of interacting with the V-ATPase complex and some members of the mTOR pathway, similar to the wild-type protein. Intriguingly, our interactomic and immunofluorescence studies showed that ΔITILELP is partially retained at the endoplasmic reticulum (ER). We proposed that the ΔITILELP mutation causes protein misfolding, ER retention and inability to be processed in the Golgi apparatus, and we demonstrated that ΔITILELP carries high-mannose glycans on all six of its remaining glycosylation sites. We found that the high turnover of ΔITILELP, because of its immature glycosylation state in combination with low transport activity, might be responsible for the phenotype observed in some patients.

Generation of PDGFR\u03b1+ Cardioblasts from Pluripotent Stem Cells

Isolating actively proliferating cardioblasts is the first crucial step for cardiac regeneration through cell implantation. However, the origin and identity of putative cardioblasts are still unclear. Here, we uncover a novel class of cardiac lineage cells, PDGFRα+Flk1− cardioblasts (PCBs), from mouse and human pluripotent stem cells induced using CsAYTE, a combination of the small molecules Cyclosporin A, the rho-associated coiled-coil kinase inhibitor Y27632, the antioxidant Trolox, and the ALK5 inhibitor EW7197. This novel population of actively proliferating cells is cardiac lineage–committed but in a morphologically and functionally immature state compared to mature cardiomyocytes. Most important, most of CsAYTE-induced PCBs spontaneously differentiated into functional αMHC+ cardiomyocytes (M+CMs) and could be a potential cellular resource for cardiac regeneration.

Solution blow spinning fibres: New immunologically inert substrates for the analysis of cell adhesion and motility.

The control of cell behaviour through material geometry is appealing as it avoids the requirement for complex chemical surface modifications. Significant advances in new technologies have been made to the development of polymeric biomaterials with controlled geometry and physico-chemical properties. Solution blow spinning technique has the advantage of ease of use allowing the production of nano or microfibres and the direct fibre deposition on any surface in situ. Yet, in spite of these advantages, very little is known about the influence of such fibres on biological functions such as immune response and cell migration. In this work, we engineered polymeric fibres composed of either pure poly(lactic acid) (PLA) or blends of PLA and polyethylene glycol (PEG) by solution blow spinning and determined their impact on dendritic cells, highly specialised cells essential for immunity and tolerance. We also determined the influence of fibres on cell adhesion and motility. Cells readily interacted with fibres resulting in an intimate contact characterised by accumulation of actin filaments and focal adhesion components at sites of cell-fibre interactions. Moreover, cells were guided along the fibres and actin and focal adhesion components showed a highly dynamic behaviour at cell-fibre interface. Remarkably, fibres did not elicit any substantial increase of activation markers and inflammatory cytokines in dendritic cells, which remained in their immature (inactive) state. Taken together, these findings will be useful for developing new biomaterials for applications in tissue engineering and regenerative medicine.

Age-Dependent Differences in Pseudorabies Virus Neuropathogenesis and Associated Cytokine Expression.

It is well known that alphaherpesvirus infections of humans and animals result in more severe clinical disease in newborns than in older individuals and that this is probably related to differences in neuropathogenesis. The underlying mechanisms, however, remain unclear. Pseudorabies virus infection of its natural host, the pig, provides a suitable infection model to study this more profoundly. We show here that the severe neurological disease observed in 2-week-old pigs does not appear to be related to a hampered innate immune response but is more likely to reflect the immature development state of the trigeminal ganglia (TG) and central nervous system (CNS) neurons, resulting in an inefficient suppression of viral replication. In 15-week-old pigs, viral replication was efficiently suppressed in the TG and CNS without induction of an extensive immune response. Furthermore, our results provide evidence that neurological disease could, at least in part, be related to viral replication and associated immunopathology in the olfactory bulb.

This Rag of Scarlet Cloth: Nathaniel Hawthorne’s Abortion

Pregnancy may be CONCEALED by an unmarried woman, and even by married ones under certain circumstances, to avoid disgrace and enable them to destroy their offspring in its mature or immature state. --Physician Theodric Beck, in his Elements of Medical Jurisprudence, 1825 What has society to do with this matter of one who was never born? --Abortionist Madame Restell, to her anonymous biographer in 1847 In his 1851 review of Scarlet Letter, Arthur Cleveland Coxe famously contends that Hawthorne's novel is at once beautiful and ambitious but that an under-tide of filth flows beneath surface of its artistry (259). Unsettled by what he sees as baffling incongruity between Hawthorne's choice of setting in the cradle of our country and topic of adultery, he asserts that Scarlet Letter encourages depravity under cover of respectability (256). Why has our author selected such a theme? Coxe asks (258); why take such a misstep (262) in representation of country? corrupting force of novel cannot be overstated, he claims, particularly because it encourages a different mode of life for women, one bound up with recent assertions of their rights at late Convention of females at Boston (262). As he builds his case against novel--its refusal to honor Puritan progenitors and its potential to lead astray daughters of America--Coxe at once incorporates genealogical terms of Anglo-American nationalism and reveals how insistently Scarlet Letter shatters them (259). Without quite being able to put his finger on it, he discerns novel as a threat to prevailing idealization of America as something born to and bequeathed by New England Puritans. Scarlet Letter indeed stages birth of nation in mother-forest on shores of Atlantic, imagining itself as new or and settlement so newly constructed, only to undermine that very idea or to name it as such: It was as if a new birth... had converted forest land, Hawthorne's narrator writes of Hester Prynne's world, calling attention to metaphor as an imposition of meaning and a falsehood (186, emphasis added). He also opens novel with reference to a portion of virgin soil where settlement's prison and its cemetery both stand, in a sly witticism that entwines womb of with its tombs (158). The spell survives, he explains in The Custom House of Salem's effect upon him, just as powerfully as if natal spell were an earthly paradise, and yet he contemplates it in very dark ways too, as a place of birth and burial where long-interred skeletons retain stain of blood on their bones (128, 126). He thus sets into motion American myth of origins--that projected [u]topia of human virtue and happiness (158)--only to cut it off. rich, longstanding literary association between textual and sexual reproduction allows him to imagine terms of a national epic, to reproduce lexicon of its idealism, and then terminate that conception's progression within his pages. With Scarlet Letter, Hawthorne hints at possibility that Anglo-America's birthright will find legitimacy under his authorship, but he engages instead in a form of abortion where no such patriotism takes hold. Hawthorne moves representation of a nation as born to or among a people into his tale about Pearl's birth, exploring what it means to see America in terms of biological gestation, as if gestation too is a form of destiny. He then runs this representation against his preference for scrapped sequences and broken chains, unpremeditated moments and latitude of speculation (204, 290). novel's main plotline itself, while luring his readers into expecting final reunion of Hester and Dimmesdale, is also severed. And it is Pearl who is asked to bear news: 'Thy mother is yonder woman with scarlet letter,' said seaman. …

Retinoic acid maintains human skeletal muscle progenitor cells in an immature state.

Muscle satellite cells are resistant to cytotoxic agents, and they express several genes that confer resistance to stress, thus allowing efficient dystrophic muscle regeneration after transplantation. However, once they are activated, this capacity to resist to aggressive agents is diminished resulting in massive death of transplanted cells. Although cell immaturity represents a survival advantage, the signalling pathways involved in the control of the immature state remain to be explored. Here, we show that incubation of human myoblasts with retinoic acid impairs skeletal muscle differentiation through activation of the retinoic-acid receptor family of nuclear receptor. Conversely, pharmacologic or genetic inactivation of endogenous retinoic-acid receptors improved myoblast differentiation. Retinoic acid inhibits the expression of early and late muscle differentiation markers and enhances the expression of myogenic specification genes, such as PAX7 and PAX3. These results suggest that the retinoic-acid-signalling pathway might maintain myoblasts in an undifferentiated/immature stage. To determine the relevance of these observations, we characterised the retinoic-acid-signalling pathways in freshly isolated satellite cells in mice and in siMYOD immature human myoblasts. Our analysis reveals that the immature state of muscle progenitors is correlated with high expression of several genes of the retinoic-acid-signalling pathway both in mice and in human. Taken together, our data provide evidences for an important role of the retinoic-acid-signalling pathway in the regulation of the immature state of muscle progenitors.

Suppression of Canine Dendritic Cell Activation/Maturation and Inflammatory Cytokine Release by Mesenchymal Stem Cells Occurs Through Multiple Distinct Biochemical Pathways.

Mesenchymal stem cells (MSC) represent a readily accessible source of cells with potent immune modulatory activity. MSC can suppress ongoing inflammatory responses by suppressing T cell function, while fewer studies have examined the impact of MSC on dendritic cell (DC) function. The dog spontaneous disease model represents an important animal model with which to evaluate the safety and effectiveness of cellular therapy with MSC. This study evaluated the effects of canine MSC on the activation and maturation of canine monocyte-derived DC, as well as mechanisms underlying these effects. Adipose-derived canine MSC were cocultured with canine DC, and the MSC effects on DC maturation and activation were assessed by flow cytometry, cytokine ELISA, and confocal microscopy. We found that canine MSC significantly suppressed lipopolysaccharide (LPS)-stimulated upregulation of DC activation markers such as major histocompatibility class II (MHCII), CD86, and CD40. Furthermore, pretreatment of MSC with interferon gamma (IFNγ) augmented this suppressive activity. IFNγ-activated MSC also significantly reduced LPS-elicited DC secretion of tumor necrosis factor alpha without reducing secretion of interleukin-10. The suppressive effect of IFNγ-treated MSC on LPS-induced DC activation was mediated by soluble factors secreted by both MSC and DC. Pathways of DC functional suppression included programmed death ligand-1 expression and secretion of nitrous oxide, prostaglandin E2, and adenosine by activated MSC. Coculture of DC with IFNγ-treated MSC maintained DC in an immature state and prolonged DC antigen uptake during LPS maturation stimulus. Taken together, canine MSC are capable of potently suppressing DC function in a potentially inflammatory microenvironment through several separate immunological pathways and confirm the potential for immune therapy with MSC in canine immune-mediated disease models.

Nestin Expression in the Adult Mouse Retina with Pharmaceutically Induced Retinal Degeneration.

The present study investigated the temporal pattern and cellular localization of nestin in the adult mouse retina with pharmaceutically induced retinal degeneration using N-methyl-N-nitrosourea (MNU). After a single intraperitoneal injection of MNU in 8-week-old C57BL/6 mice, the animals were sacrificed at 1, 3, 5, 7, and 21 days (n = 6, in each stage). The eyes were examined by means of immunohistochemical tests using nestin, ionized calcium-binding adaptor molecule (Iba-1), CD11b, F4/80, and glial fibrillary acidic protein (GFAP). Western blot analysis and manual cell counting were performed for quantification. Nestin expression was increased after MNU administration. Nestin+/Iba-1+ cells were migrated into outer nuclear layer (ONL) and peaked at day 3 post injection (PI). Nestin+/CD11b+ cells were also mainly identified in ONL at day 3 PI and peaked at day 5. Nestin+/F4/80+ cells were shown in the subretinal space and peaked at day 3 PI. Nestin+/GFAP+ cells were distinctly increased at day 1 PI and peaked at day 5 PI. The up-regulation of nestin expression after MNU administration in adult mouse retinal microglia, and monocyte/macrophage suggests that when retinal degeneration progresses, these cells may revert to a more developmentally immature state. Müller cells also showed reactive gliosis and differentiational changes.

Conversion of Terminally Committed Hepatocytes to Culturable Bipotent Progenitor Cells with Regenerative Capacity

A challenge for advancing approaches to liver regeneration is loss of functional differentiation capacity when hepatocyte progenitors are maintained in culture. Recent lineage-tracing studies have shown that mature hepatocytes (MHs) convert to an immature state during chronic liver injury, and we investigated whether this conversion could be recapitulated invitro and whether such converted cells could represent a source of expandable hepatocytes. We report that a cocktail of small molecules, Y-27632, A-83-01, and CHIR99021, can convert rat and mouse MHs invitro into proliferative bipotent cells, which we term chemically induced liver progenitors (CLiPs). CLiPs can differentiate into both MHs and biliary epithelial cells that can form functional ductal structures. CLiPs in long-term culture did not lose their proliferative capacity or their hepatic differentiation ability, and rat CLiPs were shown to extensively repopulate chronically injured liver tissue. Thus, our study advances the goals of liver regenerative medicine.

Breathing while altricial: the ontogeny of ventilatory chemosensitivity in red-winged blackbird (Agelaius phoeniceus) nestlings.

Altricial bird species, like red-winged blackbirds, hatch at an immature state of functional maturity with limited aerobic capacity and no endothermic capacity. Over the next 10-12 days in the nest, red-winged blackbirds develop increased metabolic capacity before fledging. Although ontogeny of respiration has been described in precocial birds, ontogeny of ventilatory chemosensitivity is unknown in altricial species. Here we examined developmental changes in chemosensitivity of tidal volume (Vt), breathing frequency (ƒ), minute ventilation (V̇e), and whole animal oxygen consumption (V̇o2) from hatching to just before fledging in red-winged blackbirds on days 1, 3, 5, 7, and 9 posthatching (dph) in response to hypercapnia (2 and 4% CO2) and hypoxia (15 and 10% O2). Under control conditions, there was a developmental increase in V̇e with age due to increased Vt Hypercapnic and hypoxic chemosensitivities were present as early as 1 dph. In response to hypoxia, 1, 3, and 9 dph nestlings increased V̇e at 10% O2, by increasing ƒ with some change in Vt in younger animals. In contrast to early neonatal altricial mammals, the hypoxic response of nestling red-winged blackbirds was not biphasic. In response to hypercapnia, 3 dph nestlings increased V̇e by increasing both ƒ and Vt From 5 dph on, the hypercapnic increase in V̇e was accounted for by increased Vt and not ƒ. Chemosensitivity to O2 and CO2 matures early in nestling red-winged blackbirds, well before the ability to increase V̇o2 in response to cooling, and thus does not represent a limitation to the development of endothermy.

Helical Conformation in the CA-SP1 Junction of the Immature HIV-1 Lattice Determined from Solid-State NMR of Virus-like Particles.

Maturation of HIV-1 requires disassembly of the Gag polyprotein lattice, which lines the viral membrane in the immature state, and subsequent assembly of the mature capsid protein lattice, which encloses viral RNA in the mature state. Metastability of the immature lattice has been proposed to depend on the existence of a structurally ordered, α-helical segment spanning the junction between capsid (CA) and spacer peptide 1 (SP1) subunits of Gag, a segment that is dynamically disordered in the mature capsid lattice. We report solid state nuclear magnetic resonance (ssNMR) measurements on the immature lattice in noncrystalline, spherical virus-like particles (VLPs) derived from Gag. The ssNMR data provide definitive evidence for this critical α-helical segment in the VLPs. Differences in ssNMR chemical shifts and signal intensities between immature and mature lattice assemblies also support a major rearrangement of intermolecular interactions in the maturation process, consistent with recent models from electron cryomicroscopy and X-ray crystallography.

Imaging Cell Viability on Non-transparent Scaffolds &amp;#8212; Using the Example of a Novel Knitted Titanium Implant

Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in nucleus pulposus (NP) extrusion leads to intervertebral disc destruction. Currently available surgical treatments reduce the pain but do not restore the mechanical functionality of the spine. In order to preserve mechanical features of the spine, total disc or nucleus replacement thus became a wide interest. However, this arthroplasty era is still in an immature state, since none of the existing products have been clinically evaluated. This study intends to test the biocompatibility of a novel nucleus implant made of knitted titanium wires. Despite all mechanical advantages, the material has its limits for conventional optical analysis as the resulting implant is non-transparent. Here we present a strategy that describes in vitro visualization, tracking and viability testing of osteochondro-progenitor cells on the scaffold. This protocol can be used to visualize the efficiency of the cleaning protocol as well as to investigate the biocompatibility of these and other non-transparent scaffolds. Furthermore, this protocol can be used to show adherence pattern of cells as well as cell viability and proliferation rates on/in the scaffold. This in vitro biocompatibility testing assay provides a propitious tool to analyze cell-material interaction in non-transparent and opaque scaffolds.