Understanding Pseudomonas aeruginosa-Host Interactions: The Ongoing Quest for an Efficacious Vaccine.

Pseudomonas aeruginosa is a leading cause of chronic respiratory infections in people with cystic fibrosis (CF), bronchiectasis or chronic obstructive pulmonary disease (COPD), and acute infections in immunocompromised individuals. The adaptability of this opportunistic pathogen has hampered the development of antimicrobial therapies, and consequently, it remains a major threat to public health. Due to its antimicrobial resistance, vaccines represent an alternative strategy to tackle the pathogen, yet despite over 50 years of research on anti-Pseudomonas vaccines, no vaccine has been licensed. Nevertheless, there have been many advances in this field, including a better understanding of the host immune response and the biology of P. aeruginosa. Multiple antigens and adjuvants have been investigated with varying results. Although the most effective protective response remains to be established, it is clear that a polarised Th2 response is sub-optimal, and a mixed Th1/Th2 or Th1/Th17 response appears beneficial. This comprehensive review collates the current understanding of the complexities of P. aeruginosa-host interactions and its implication in vaccine design, with a view to understanding the current state of Pseudomonal vaccine development and the direction of future efforts. It highlights the importance of the incorporation of appropriate adjuvants to the protective antigen to yield optimal protection.

Antigen-Conjugated Silica Solid Sphere as Nanovaccine for Cancer Immunotherapy.

BACKGROUND: Nanocarriers could deliver significantly higher amounts of antigen to antigen-presenting cells (APCs), which have great potential to stimulate humoral and cellular response in cancer immunotherapy. Thereafter, silica solid nanosphere (SiO2) was prepared, and a model antigen (ovalbumin, OVA) was covalently conjugated on the surface of SiO2 to form nanovaccine (OVA@SiO2). And the application of OVA@SiO2 for cancer immunotherapy was evaluated. MATERIALS AND METHODS: SiO2 solid nanosphere was prepared by the Stöber method, then successively aminated by aminopropyltriethoxysilane and activated with glutaraldehyde. OVA was covalently conjugated on the surface of activated SiO2 to obtain nanovaccine (OVA@SiO2). Dynamic light scattering, scanning electron microscope, and transmission electron microscope were conducted to identify the size distribution, zeta potential and morphology of OVA@SiO2. The OVA loading capacity was investigated by varying glutaraldehyde concentration. The biocompatibility of OVA@SiO2 to DC2.4 and RAW246.7 cells was evaluated by a Cell Counting Kit-8 assay. The uptake of OVA@SiO2 by DC2.4 and its internalization pathway were evaluated in the absence or presence of different inhibitors. The activation and maturation of bone marrow-derived DC cells by OVA@SiO2 were also investigated. Finally, the in vivo transport of OVA@SiO2 and its toxicity to organs were appraised. RESULTS: All results indicated the successful covalent conjugation of OVA on the surface of SiO2. The as-prepared OVA@SiO2 possessed high antigen loading capacity, which had good biocompatibility to APCs and major organs. Besides, OVA@SiO2 facilitated antigen uptake by DC2.4 cells and its cytosolic release. Noteworthily, OVA@SiO2 significantly promoted the maturation of dendritic cells and up-regulation of cytokine secretion by co-administration of adjuvant CpG-ODN. CONCLUSION: The as-prepared SiO2 shows promising potential for use as an antigen delivery carrier.

Photothermal Therapy Promotes Tumor Infiltration and Antitumor Activity of CAR T Cells.

Chimeric antigen receptor (CAR)-redirected T lymphocytes (CAR T cells) show modest therapeutic efficacy in solid tumors. The desmoplastic structure of the tumor and the immunosuppressive tumor microenvironment usually account for the reduced efficacy of CAR T cells in solid tumors. Mild hyperthermia of the tumor reduces its compact structure and interstitial fluid pressure, increases blood perfusion, releases antigens, and promotes the recruitment of endogenous immune cells. Therefore, the combination of mild hyperthermia with the adoptive transfer of CAR T cells can potentially increase the therapeutic index of these cells in solid tumors. It is found that the chondroitin sulfate proteoglycan-4 (CSPG4)-specific CAR T cells infused in Nod scid gamma mice engrafted with the human melanoma WM115 cell line have superior antitumor activity after photothermal ablation of the tumor. The findings suggest that photothermal therapy facilitates the accumulation and effector function of CAR T cells within solid tumors.

Isolation, cultivation, and characterization of primary bovine cochlear pericytes: A new in vitro model of stria vascularis.

The study of strial pericytes has gained great interest as they are pivotal for the physiology of stria vascularis. To provide an easily accessible in vitro model, here we described a growth medium-based approach to obtain and cultivate primary bovine cochlear pericytes (BCP) from the stria vascularis of explanted bovine cochleae. We obtained high-quality pericytes in 8-10 days with a > 90% purity after the second passage. Immunocytochemical analysis showed a homogeneous population of cells expressing typical pericyte markers, such as neural/glial antigen 2 (NG2), platelet-derived growth factor receptorß (PDGFRß), α-smooth muscle actin (α-SMA), and negative for the endothelial marker von Willebrand factor. When challenged with tumor necrosis factor or lipopolysaccharide, BCP changed their shape, similarly to human retinal pericytes (HRPC). The sensitivity of BCP to ototoxic drugs was evaluated by challenging with cisplatin or gentamicin for 48 hr. Compared to human retinal endothelial cells and HRPC, cell viability of BCP was significantly lower ( p < 0.05) after the treatment with gentamicin or cisplatin. These data indicate that our protocol provides a simple and reliable method to obtain highly pure strial BCP. Furthermore, BCP are suitable to assess the safety profile of molecules which supposedly exert ototoxic activity, and may represent a valid alternative to in vivo tests.

Fas receptor induces apoptosis of synovial bone and cartilage progenitor populations and promotes bone loss in antigen-induced arthritis.

Rheumatoid arthritis (RA) is an inflammatory joint disease that eventually leads to permanent bone and cartilage destruction. Fas has already been established as the regulator of inflammation in RA, but its role in bone formation under arthritic conditions is not completely defined. The aim of this study was to assess the effect of Fas inactivation on the bone damage during murine antigen-induced arthritis. Subchondral bone of wild-type (WT) and Fas-knockout (Fas-/-) mice was evaluated by histomorphometry and microcomputerized tomography. Proportions of synovial bone and cartilage progenitors were assessed by flow cytometry. Synovial bone and cartilage progenitors were purified by fluorescence-activated cell sorting and expression of Fas and Fas-induced apoptosis were analyzed in vitro. Results showed that Fas-/- mice developed attenuated arthritis characterized by preserved epiphyseal bone and cartilage. A proportion of the earliest CD200+ bone and cartilage progenitors was reduced in WT mice with arthritis and was unaltered in Fas-/- mice. During osteoblastic differentiation in vitro, CD200+ cells express the highest levels of Fas and are removed by Fas ligation. These results suggest that Fas-induced apoptosis of early CD200+ osteoprogenitor population represents potential mechanism underlying the impaired bone formation in arthritis, so their preservation may represent the bone-protective mechanism during arthritis.-Lazic Mosler, E., Lukac, N., Flegar, D., Fadljevic, M., Radanovic, I., Cvija, H., Kelava, T., Ivcevic, S., Sucur, A., Markotic, A., Katavic, V., Marusic, A., Grcevic, D., Kovacic, N. Fas receptor induces apoptosis of synovial bone and cartilage progenitor populations and promotes bone loss in antigen-induced arthritis.

Cell and fluid sampling microneedle patches for monitoring skin-resident immunity.

Important cell populations reside within tissues and are not accessed by traditional blood draws used to monitor the immune system. To address this issue at an essential barrier tissue, the skin, we created a microneedle-based technology for longitudinal sampling of cells and interstitial fluid, enabling minimally invasive parallel monitoring of immune responses. Solid microneedle projections were coated by a cross-linked biocompatible polymer, which swells upon skin insertion, forming a porous matrix for local leukocyte infiltration. By embedding molecular adjuvants and specific antigens encapsulated in nanocapsules within the hydrogel coating, antigen-specific lymphocytes can be enriched in the recovered cell population, allowing for subsequent detailed phenotypic and functional analysis. We demonstrate this approach in mice immunized with a model protein antigen or infected in the skin with vaccinia virus. After vaccination or infection, sampling microneedles allowed tissue-resident memory T cells (TRMs) to be longitudinally monitored in the skin for many months, during which time the antigen-specific T cell population in systemic circulation contracted to low or undetectable counts. Sampling microneedles did not change the immune status of naïve or antigen-exposed animals. We also validated the ability of cell sampling using human skin samples. This approach may be useful in vaccines and immunotherapies to temporally query TRM populations or as a diagnostic platform to sample for biomarkers in chronic inflammatory and autoimmune disorders, allowing information previously accessible only via invasive biopsies to be obtained in a minimally invasive manner from the skin or other mucosal tissues.

Evaluating Antigen-Specific IgE Using the Rat Basophil Leukemia Cell (RBL) Assay.

Allergic diseases (atopy) include asthma, allergic rhinitis, conjunctivitis, and allergic sinusitis. It is estimated that up to 90% of asthmatics are atopic and have an allergy trigger for asthmatic episodes. In order to assess the risk of allergy induction associated with inhalation exposure, animal models of protein allergy have been developed. These models have been used both to identify proteins as allergens and to assess their relative potency. Often these research situations include allergens that are not well characterized or are unknown. In these situations, specific allergens are not available to be evaluated by more well-known assays (such as ELISAs), and developing a specific assay to evaluate an extract or mixture for an unknown or potential allergen is very time consuming and generally requires purified antigen/allergen. Additionally, when the comparison of the relative potency of multiple extracts is of interest, a common/generic platform is necessary. A more generic method, the rat basophil leukemia cell assay (RBL assay), has been developed which provides insight into the allergenicity of extracts and mixtures as well as providing a common platform for relative potency comparison between/among these complex allergen sources.

Quantitative Analysis of Vaccine Antigen Adsorption to Aluminum Adjuvant Using an Automated High-Throughput Method.

Aluminum-containing adjuvants have been widely used in vaccine formulations to safely and effectively potentiate the immune response. The examination of the extent of antigen adsorption to aluminum adjuvant is always evaluated during the development of aluminum adjuvant containing vaccines. A rapid, automated, high-throughput assay was developed to measure antigen adsorption in a 96-well plate format using a TECAN Freedom EVO® (TECAN). The antigen adsorption levels at a constant adjuvant concentration for each sample were accurately measured at 12 antigen/adjuvant (w/w) formulation ratios. These measurements were done at aluminum adjuvant concentrations similar to normal vaccine formulations, unlike previous non-automated and automated adjuvant adsorption studies. Two high-sensitivity analytical methods were used to detect the non-absorbed antigens. The antigen-to-adjuvant adsorption curves were fit to a simple Langmuir adsorption model for quantitatively analyzing the antigen to the adjuvant adsorption level and strength. The interaction of two model antigens, bovine serum albumin and lysozyme, with three types of aluminum adjuvant, were quantitatively analyzed in this report. Automated, high-throughput methodologies combined with sensitive analytical methods are useful for accelerating practical vaccine formulation development.LAY ABSTRACT: Vaccines are probably the most effective public health method to prevent epidemics of many infectious diseases. Many of the most effective vaccines contain aluminum adjuvant. This report describes novel technology that can be used to better optimize the efficacy and stability of aluminum adjuvant-containing vaccines.

Understanding the Effect of Surface Chemistry of Mesoporous Silica Nanorods on Their Vaccine Adjuvant Potency.

Mesoporous silica nanoparticles are reported as adjuvants in nanovaccines in generating robust antigen-specific immunity. However, the effect of surface chemistry in initiating and modulating the immune response remains largely unexplored. In this study, mesoporous silica nanorods (MSNRs) are modified with NH2 and C18 groups to investigate the influence of surface functional groups (OH, NH2 , and C18 ) on their adjuvant efficacy. It is found that compared to OH and NH2 groups, the hydrophobic C18 modification significantly enhances antigen uptake by antigen presenting cells and endosomal-lysosomal escape in vitro, dendritic cells, and macrophages maturation ex vivo, and elicits secretion of interferon-γ level and antibody response in immunized mice. Moreover, bare MSNR and MSNRNH2 exhibit T-helper 2 biased immune response, while MSNRC18 shows a T-helper 1 biased immune response. These findings suggest that the surface chemistry of nanostructured adjuvants has profound impact on the immune response, which provides useful guidance for the design of effective nanomaterial based vaccines.

Mechanisms of Antigen Adsorption Onto an Aluminum-Hydroxide Adjuvant Evaluated by High-Throughput Screening.

The adsorption mechanism of antigen on aluminum adjuvant can affect antigen elution at the injection site and hence the immune response. Our aim was to evaluate adsorption onto aluminum hydroxide (AH) by ligand exchange and electrostatic interactions of model proteins and antigens, bovine serum albumin (BSA), ß-casein, ovalbumin (OVA), hepatitis B surface antigen, and tetanus toxin (TT). A high-throughput screening platform was developed to measure adsorption isotherms in the presence of electrolytes and ligand exchange by a fluorescence-spectroscopy method that detects the catalysis of 6,8-difluoro-4-methylumbelliferyl phosphate by free hydroxyl groups on AH. BSA adsorption depended on predominant electrostatic interactions. Ligand exchange contributes to the adsorption of ß-casein, OVA, hepatitis B surface antigen, and TT onto AH. Based on relative surface phosphophilicity and adsorption isotherms in the presence of phosphate and fluoride, the capacities of the proteins to interact with AH by ligand exchange followed the trend: OVA < ß-casein < BSA < TT. This could be explained by both the content of ligands available in the protein structure for ligand exchange and the antigen's molecular weight. The high-throughput screening platform can be used to better understand the contributions of ligand exchange and electrostatic attractions governing the interactions between an antigen adsorbed onto aluminum-containing adjuvant.

Armillaridin, a Honey Medicinal Mushroom, Armillaria mellea (Higher Basidiomycetes) Component, Inhibits Differentiation and Activation of Human Macrophages.

Armillaridin (AM) is an aromatic ester compound isolated from honey medicinal mushroom, Armillaria mellea, which has anti-cancer potential. This study was designed to examine the effects of AM on differentiation and activation macrophages, the major ontogeny of innate immunity. Macrophages were derived from CD14+ monocytes which were sorted from human peripheral blood mononuclear cells. Cell viability was assessed by trypan blue exclusion test. Cells were stained with Liu's dye for observation of morphology. Expression of surface antigens was examined by flow cytometric analysis. Phagocytosis and generation of reactive oxygen species (ROS), as functional assays, were evaluated by counting engulfed yeasts and DCFH-DA reaction. The viability of macrophages was not significantly reduced by AM. AM at nontoxic concentrations markedly increased cytoplasmic vacuoles. The expression of surface CD14, CD16, CD36, and HLA-DR was suppressed. The phagocytosis function, but not ROS production, of macrophages was inhibited by AM. Armillaridin could inhibit the differentiation and activation of human macrophages. It may have potential to be developed as a biological response modifier for inflammatory diseases.

Mesoporous silica particles potentiate antigen-specific T-cell responses.

AIM: To study the adjuvant effect of mesoporous silica particles and their capability of modifying an already existing allergic Th2-like immune response. MATERIALS & METHODS: The adjuvant effect of Santa Barbara Amorphous-15 (SBA-15) mesoporous silica particles was studied in an antigen-specific ovalbumin (OVA) system in vitro and in vivo. The capacity of the OVA-loaded SBA-15 particles (SBA-15-OVA) to modify an existing immune response was assessed in a murine allergy model. RESULTS: SBA-15-OVA induced significantly stronger OVA-specific splenocyte proliferation compared with OVA alone. Significantly higher IFN-γ production was observed in ex vivo OVA-stimulated splenocytes from SBA-15-OVA-immunized mice compared with mice injected with only SBA-15 or OVA. Treatment of OVA-sensitized mice with SBA-15-OVA modified the immune response with significantly lower serum levels of OVA-specific IgE and higher IgG levels compared with the alum-OVA-treated group. CONCLUSION: The results are promising for the continued development of mesoporous silica materials for therapeutic applications.

Fever-range whole-body heat treatment stimulates antigen-specific T-cell responses in humans.

Increase in body temperature has been thought to play an important role in the regulation of immune responses, although its precise mechanisms are still under investigation. Here, we examined the effects of physiologically relevant thermal stress on the cytokine production from human peripheral T cells. Volunteers were heated using a whole-body hyperthermia device, the rectal temperature was maintained above 38.5 °C for more than 60 min, and peripheral blood mononuclear cells (PBMCs) were obtained before and after the treatment. When T cells were stimulated with anti-CD3/CD28 antibodies, marked increases in the production of interferon-γ (IFN-γ) and interleukin-2 were observed in PBMCs prepared immediately after and 24h after the treatment. Similarly, enhanced production of IFN-γ in response to the tuberculin purified protein derivative or antigenic viral peptides was also observed immediately after and 24h after the treatment. Fluorescence photo-bleaching analyses showed heat-induced increase of membrane fluidity in T cells, which probably enables them to induce rapid and efficient cluster formation of molecules involved in antigen recognition and signal transduction for T-cell stimulation. We concluded that physiologically relevant thermal stress could efficiently modify T-cell responsiveness to various stimuli, including enhanced responses to specific antigens.

Changes in pericytic expression of NG2 and PDGFRB and vascular permeability in the sensory circumventricular organs of adult mouse by osmotic stimulation.

The blood-brain barrier (BBB) is a barrier that prevents free access of blood-derived substances to the brain through the tight junctions and maintains a specialized brain environment. Circumventricular organs (CVOs) lack the typical BBB. The fenestrated vasculature of the sensory CVOs, including the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO) and area postrema (AP), allows parenchyma cells to sense a variety of blood-derived information, including osmotic ones. In the present study, we utilized immunohistochemistry to examine changes in the expression of NG2 and platelet-derived growth factor receptor beta (PDGFRB) in the OVLT, SFO and AP of adult mice during chronic osmotic stimulation. The expression of NG2 and PDGFRB was remarkably prominent in pericytes, although these angiogenesis-associated proteins are highly expressed at pericytes of developing immature vasculature. The chronic salt loading prominently increased the expression of NG2 in the OVLT and SFO and that of PDGFRB in the OVLT, SFO and AP. The vascular permeability of low-molecular-mass tracer fluorescein isothiocyanate was increased significantly by chronic salt loading in the OVLT and SFO but not AP. In conclusion, the present study demonstrates changes in pericyte expression of NG2 and PDGFRB and vascular permeability in the sensory CVOs by chronic osmotic stimulation, indicating active participation of the vascular system in osmotic homeostasis.

Efeitos de compostos naturais, sintéticos e da fototerapia antifúngica sobre Candida tropicalis resistente ao fluconazol / Effects of natural compounds, synthetic and herbal medicine on Candida tropicalis antifungal fluconazole-resistant

A candidíase é uma infecção oportunista provocada por diversas espécies de fungos do gênero Candida, frequentemente encontrados integrando a microbiota, da superfície cutânea, no trato gastrointestinal e cavidades mucosas do ser humano desde o seu nascimento. A incidência das infecções fúngicas sistêmicas têm aumentado consideravelmente nas últimas décadas em função do grande número de pacientes com SIDA, a grande quantidade de transplantes e condições crônicas como o câncer, a terapia prolongada com imunossupressores e o uso de agentes corticosteroides. Além disso, a exposição prolongada aos antifúngicos azólicos promove a seleção de patógenos resistentes. No presente estudo avaliou-se a atividade antifúngica do complexo Rutênio-pirocatecol (RPC) frente a um isolado clinico de Candida tropicalis resistente ao fluconazol. A metodologia empregada para os testes de susceptibilidade foi de acordo com o documento M27-A3 do National Committee for Clinical Laboratory Standards (NCCLS, 2008). Esplenócitos de camundongos Balb/c foram obtidos de forma asséptica para avaliar a citotoxicidade do composto para células de mamíferos. O estresse oxidativo promovido pelo composto foi avaliado através da reação ao ácido tiobarbitúrico (TBARS) e ensaios de fluorescência com a sonda diclorodihidrofluoresceína diacetato (DCFH2DA). O Calcofluor White foi empregado para avaliar a integridade da parede celular. A análise ultraestrutural foi realizada através da microscopia eletrônica de varredura e transmissão. Os resultados encontrados para os testes de atividade antifúngica foram analisados através do teste estatístico ANOVA e pós-teste Dunnett. Os resultados encontrados para os testes de atividade antifúngica do RPC mostraram uma Concentração Inibitória de 50% (IC50) de 20,3 μM, enquanto em esplesnócitos a concentração efetiva de 50% foi de 325 μM mostrando um índice de seletividade igual a 16...

Candidiasis is an opportunistic infection caused by several species of fungi of the genus Candida, often found is the microbiota, on the skin, gastrointestinal tract and mucous cavities of the human beings birth. The incidence of systemic fungal infections have increased considerably in recent decades due to the large number of AIDS patients, the large number of transplants and chronic conditions such as cancer, prolonged therapy promotes the selection of resistant pathogen with immunosuppressant and corticosteroid agents. Also prolonged exposure azole antifungals to make them strong candidates for patients resistance. In the present study we evaluated the antifungal activity of Ruthenium-pyrocatechol complex (RPC) against a clinical isolate of Candida tropicalis resistant to fluconazole. The methodology for susceptibility testing was in accordance with the M27-A3 document of there National Committee for Clinical Laboratory Standards (NCCLS, 2008). Splenocytes from Balb/c mice were obtained aseptically to evaluate the cytotoxicity of the compound to mammalian cells. Oxidative stress caused by the compound was assessed by reaction to thiobarbituric acid (TBARS) and fluorescence assays with the probe diclorodihidrofluoresceína diacetate (DCFH2DA). The Calcofluor White was used to evaluate the integrity of the cell wall. The ultrastructural analysis was performed by scanning and transmission electron microscopy. The results for the antifungal activity tests were analyzed using ANOVA and pos-test Dunnett test statistic. The results for the tests of antifungal activity of the RPC showed a 50% inhibitory concentration (IC50) of 20.3 μM while in splenocytes the 50% effective concentration was 325 μM showing a selectivity index of 16...

Evidence for NG2-glia derived, adult-born functional neurons in the hypothalamus.

Accumulating evidence suggests that the adult murine hypothalamus, a control site of several fundamental homeostatic processes, has neurogenic capacity. Correspondingly, the adult hypothalamus exhibits considerable cell proliferation that is ongoing even in the absence of external stimuli, and some of the newborn cells have been shown to mature into cells that express neuronal fate markers. However, the identity and characteristics of proliferating cells within the hypothalamic parenchyma have yet to be thoroughly investigated. Here we show that a subset of NG2-glia distributed throughout the mediobasal hypothalamus are proliferative and express the stem cell marker Sox2. We tracked the constitutive differentiation of hypothalamic NG2-glia by employing genetic fate mapping based on inducible Cre recombinase expression under the control of the NG2 promoter, demonstrating that adult hypothalamic NG2-glia give rise to substantial numbers of APC+ oligodendrocytes and a smaller population of HuC/D+ or NeuN+ neurons. Labelling with the cell proliferation marker BrdU confirmed that some NG2-derived neurons have proliferated shortly before differentiation. Furthermore, patch-clamp electrophysiology revealed that some NG2-derived cells display an immature neuronal phenotype and appear to receive synaptic input indicative of their electrical integration in local hypothalamic circuits. Together, our studies show that hypothalamic NG2-glia are able to take on neuronal fates and mature into functional neurons, indicating that NG2-glia contribute to the neurogenic capacity of the adult hypothalamus.

Biodistribution of vaccines comprised of hydrophobically-modified poly(γ-glutamic acid) nanoparticles and antigen proteins using fluorescence imaging.

Fluorophores-modified nanoparticles comprised of poly(γ-glutamic acid)-phenylalanine (γ-PGA-Phe-633) and ovalbumin (OVA-750) termed NPs-633/OVA-750 were prepared to assess their biodistribution using an in vivo fluorescence imager. Dynamic light scattering measurements indicated that NPs-633/OVA-750 were about 200nm in diameter. The release of encapsulated OVA from NPs-633 in PBS was negligible (∼10%) for a week. When subcutaneously injected, the localization period of OVA-750-encapsulated into NPs-633 at the site of injection (SOI) was much longer than that of free OVA-750, but was shorter as compared to a mixture with aluminum hydroxide. The NPs-633 disappeared at the SOI and major organs within 1month after administration. Moreover, intravenously and intraperitoneally administered NPs-633 were mainly observed at the liver, and there was more rapid clearance from all organs as compared with non-biodegradable NPs. These fast clearance and degradation characteristics of γ-PGA-Phe NPs will be important not only for avoiding undesired adverse effects, but also for inducing a strong vaccine effect.

Extensive regenerative plasticity among adult NG2-glia populations is exclusively based on self-renewal.

NG2-glia are known to proliferate in the adult brain, however the extent of their mitotic and regenerative capacity and particularly their adult origin is uncertain. By employing a paradigm of mitotic blockade in conjunction with genetic fate tracing we demonstrate that intracerebroventricular mitotic blocker infusion leads to wide-spread and complete ablation of NG2-glial cells in the hypothalamus and other periventricular brain regions. However, despite the extensive glia loss, parenchymal NG2-glia coverage is fully restored to pretreatment levels within two weeks. We further reveal that in response to mitotic blocker treatment, NG2-glia bordering the ablated territories start to express the stem cell marker nestin, divide and migrate to replace the lost cells. Importantly, the migration front of repopulating NG2-glia invariably proceeds from the distal parenchyma towards the ventricles, ruling out contributions of the subventricular zone neurogenic niche or the corresponding area of the third ventricle as source of new NG2-glia. NG2-CreER-based fate tracing further substantiates that NG2-glia which have been spared from mitotic blockade are the sole source of regenerating NG2-glia. Collectively, our data reveals that all adult NG2-glia retain the ability to divide and that they are capable of fully restoring parenchymal NG2-glia coverage after wide-spread NG2 cell loss, indicating complete self-sufficiency in maintaining NG2-glia population levels in the adult brain.

Hunger-promoting hypothalamic neurons modulate effector and regulatory T-cell responses.

Whole-body energy metabolism is regulated by the hypothalamus and has an impact on diverse tissue functions. Here we show that selective knockdown of Sirtuin 1 Sirt1 in hypothalamic Agouti-related peptide-expressing neurons, which renders these cells less responsive to cues of low energy availability, significantly promotes CD4(+) T-cell activation by increasing production of T helper 1 and 17 proinflammatory cytokines via mediation of the sympathetic nervous system. These phenomena were associated with an impaired thymic generation of forkhead box P3 (FoxP3(+)) naturally occurring regulatory T cells and their reduced suppressive capacity in the periphery, which resulted in increased delayed-type hypersensitivity responses and autoimmune disease susceptibility in mice. These observations unmask a previously unsuspected role of hypothalamic feeding circuits in the regulation of adaptive immune response.

Neural and oligodendrocyte progenitor cells: transferrin effects on cell proliferation.

NSC (neural stem cells)/NPC (neural progenitor cells) are multipotent and self-renew throughout adulthood in the SVZ (subventricular zone) of the mammalian CNS (central nervous system). These cells are considered interesting targets for CNS neurodegenerative disorder cell therapies, and understanding their behaviour in vitro is crucial if they are to be cultured prior to transplantation. We cultured the SVZ tissue belonging to newborn rats under the form of NS (neurospheres) to evaluate the effects of Tf (transferrin) on cell proliferation. The NS were heterogeneous in terms of the NSC/NPC markers GFAP (glial fibrillary acidic protein), Nestin and Sox2 and the OL (oligodendrocyte) progenitor markers NG2 (nerve/glia antigen 2) and PDGFRα (platelet-derived growth factor receptor α). The results of this study indicate that aTf (apoTransferrin) is able to increase cell proliferation of SVZ-derived cells in vitro, and that these effects were mediated at least in part by the TfRc1 (Tf receptor 1). Since OPCs (oligodendrocyte progenitor cells) represent a significant proportion of the proliferating cells in the SVZ-derived primary cultures, we used the immature OL cell line N20.1 to show that Tf was able to augment the proliferation rate of OPC, either by adding aTf to the culture medium or by overexpressing rat Tf in situ. The culture medium supplemented with ferric iron, together with aTf, increased the DNA content, while ferrous iron did not. The present work provides data that could have a potential application in human cell replacement therapies for neurodegenerative disease and/or CNS injury that require the use of in vitro amplified NPCs.