A step forwards in immunomodulation and immunetolerance knowledge. HLA-G expression in co-cultures of peripheral blood mononuclear cells and stem cells after in vitro NGAL stimulation

NGAL (Neutrophil Gelatinase-associated Lipocalin ) is a protein of lipocalin superfamily. Recent literature focused on its biomarkers function in several pathological condition (acute and chronic kidney damage, autoimmune disease, malignancy). NGAL biological role is not well elucidated. Several are the demonstration of its bacteriostatic role. Recent papers have indeed highlight NGAL role in NFkB modulation. The aim of this study is to understand whether NGAL may exert a role in the activation (modulation) of T cell response through the regulation of HLA-G complex, a mediator of tolerance. From 8 healthy donors we obtained peripheral blood mononuclear cells (PBMCs) and we isolated by centrifugation on a Ficoll gradient. Cells were then treated with four concentrations of NGAL (40-320 ng/ml) with or without iron. We performed flow cytometry analysis and ELISA test. NGAL increased the HLA-G expression on CD4+ T cells, with an increasing corresponding to the dose. Iron effect is not of unique interpretation. NGAL adiction affects regulatory T cells increasing in vitro expansion of CD4+ CD25+ FoxP3+ cells. Neutralizing antibody against NGAL decreased HLA-G expression and reduced significantly CD4+ CD25+ FoxP3+ cells percentage. In conclusion, we provided in vitro evidence of NGAL involvement in cellular immunity. The potential role of NGAL as an immunomodulatory molecule has been evaluated: it has been shown that NGAL plays a pivotal role in the induction of immune tolerance up regulating HLA-G and T regulatory cells expression in healthy donors. As potential future scenario we highlight the in vivo role of NGAL in immunology and immunomodulation, and its possible relationship with immunosuppressive therapy efficacy, tolerance induction in transplant patients, and/or in other immunological disorders.

Use of a 3D perfusion bioreactor with osteoblasts and osteoblast/endothelial cell co-cultures to improve tissue-engineered bone

The delivery of oxygen, nutrients, and the removal of waste are essential for cellular survival. Culture systems for 3D bone tissue engineering have addressed this issue by utilizing perfusion flow bioreactors that stimulate osteogenic activity through the delivery of oxygen and nutrients by low-shear fluid flow. It is also well established that bone responds to mechanical stimulation, but may desensitize under continuous loading. While perfusion flow and mechanical stimulation are used to increase cellular survival in vitro, 3D tissue-engineered constructs face additional limitations upon in vivo implantation. As it requires significant amounts of time for vascular infiltration by the host, implants are subject to an increased risk of necrosis. One solution is to introduce tissue-engineered bone that has been pre-vascularized through the co-culture of osteoblasts and endothelial cells on 3D constructs. It is unclear from previous studies: 1) how 3D bone tissue constructs will respond to partitioned mechanical stimulation, 2) how gene expression compares in 2D and in 3D, 3) how co-cultures will affect osteoblast activity, and 4) how perfusion flow will affect co-cultures of osteoblasts and endothelial cells. We have used an integrated approach to address these questions by utilizing mechanical stimulation, perfusion flow, and a co-culture technique to increase the success of 3D bone tissue engineering. We measured gene expression of several osteogenic and angiogenic genes in both 2D and 3D (static culture and mechanical stimulation), as well as in 3D cultures subjected to perfusion flow, mechanical stimulation and partitioned mechanical stimulation. Finally, we co-cultured osteoblasts and endothelial cells on 3D scaffolds and subjected them to long-term incubation in either static culture or under perfusion flow to determine changes in gene expression as well as histological measures of osteogenic and angiogenic activity. We discovered that 2D and 3D osteoblast cultures react differently to shear stress, and that partitioning mechanical stimulation does not affect gene expression in our model. Furthermore, our results suggest that perfusion flow may rescue 3D tissue-engineered constructs from hypoxic-like conditions by reducing hypoxia-specific gene expression and increasing histological indices of both osteogenic and angiogenic activity. Future research to elucidate the mechanisms behind these results may contribute to a more mature bone-like structure that integrates more quickly into host tissue, increasing the potential of bone tissue engineering.

Discovery of "isolated" co-moving T Tauri stars in Cepheus

Context. During the course of a large spectroscopic survey of X-ray active late-type stars in the solar neighbourhood, we discovered four lithium-rich stars packed within just a few degrees on the sky. Although located in a sky area rich in CO molecular regions and dark clouds, the Cepheus-Cassiopeia complex, these very young stars are projected several degrees away from clouds in front of an area void of interstellar matter. As such, they are very good "isolated" T Tauri star candidates. Aims. We present optical observations of these stars conducted with 1-2 m class telescopes. We acquired high-resolution optical spectra as well as photometric data allowing us to investigate in detail their nature and physical parameters with the aim of testing the "runaway" and "in-situ" formation scenarios. Their kinematical properties are also analyzed to investigate their possible connection to already known stellar kinematic groups. Methods. We use the cross-correlation technique and other tools developed by us to derive accurate radial and rotational velocities and perform an automatic spectral classification. The spectral subtraction technique is used to infer chromospheric activity level in the Hα line core and clean the spectra of photospheric lines before measuring the equivalent width of the lithium absorption line. Results. Both physical (lithium content, chromospheric, and coronal activities) and kinematical indicators show that all stars are very young, with ages probably in the range 10-30 Myr. In particular, the spectral energy distribution of TYC4496-780-1 displays a strong near-and far-infrared excess, typical of T Tauri stars still surrounded by an accretion disc. They also share the same Galactic motion, proving that they form a homogeneous moving group of stars with the same origin. Conclusions. The most plausible explanation of how these "isolated" T Tauri stars formed is the "in-situ" model, although accurate distances are needed to clarify their connection with the Cepheus-Cassiopeia complex. The discovery of this loose association of "isolated" T Tauri stars can help to shed light on atypical formation processes of stars and planets in low-mass clouds.

Bioactivity of Ruta graveolens and Satureja montana Essential Oils on Solanum tuberosum Hairy Roots and Solanum tuberosum Hairy Roots with Meloidogyne chitwoodi Co-cultures

As a nematotoxics screening biotechnological system, Solanum tuberosum hairy roots (StHR) and S. tuberosum hairy roots with Meloidogyne chitwoodi co-cultures (StHR/CRKN) were evaluated, with and without the addition of the essential oils (EOs) of Satureja montana and Ruta graveolens. EOs nematotoxic and phytotoxic effects were followed weekly by evaluating nematode population density in the co-cultures as well as growth and volatile profiles of both in vitro cultures types. Growth, measured by the dissimilation method and by fresh and dry weight determination, was inhibited after EO addition. Nematode population increased in control cultures, while in EO-added cultures numbers were kept stable. In addition to each of the EOs main components, and in vitro cultures constitutive volatiles, new volatiles were detected by gas chromatography and gas chromatography coupled to mass spectrometry in both culture types. StHR with CRKN co-cultures showed to be suitable for preliminary assessment of nematotoxic EOs.

Allelopathic interactions between the macroalga Ulva pertusa and eight microalgal species

HETEROSIGMA-AKASHIWO RAPHIDOPHYCEAE; CENTRAL VENICE LAGOON; ALEXANDRIUM-TAMARENSE; RED-TIDE; COASTAL LAGOONS; PHYTOPLANKTON; GROWTH; BAY; DINOFLAGELLATE; COMPETITION

Investigation of a new acetogen isolated from an enrichment of the tammar wallaby forestomach

Background: Forestomach fermentation in Australian marsupials such as wallabies and kangaroos, though analogous to rumen fermentation, results in lower methane emissions. Insights into hydrogenotrophy in these systems could help in devising strategies to reduce ruminal methanogenesis. Reductive acetogenesis may be a significant hydrogen sink in these systems and previous molecular analyses have revealed a novel diversity of putative acetogens in the tammar wallaby forestomach.Results: Methanogen-inhibited enrichment cultures prepared from tammar wallaby forestomach contents consumed hydrogen and produced primarily acetate. Functional gene (formyltetrahydrofolate synthetase and acetyl-CoA synthase) analyses revealed a restricted diversity of Clostridiales species as the putative acetogens in the cultures. A new acetogen (growth on H-2/CO2 with acetate as primary end product) designated isolate TWA4, was obtained from the cultures. Isolate TWA4 classified within the Lachnospiraceae and demonstrated > 97% rrs identity to previously isolated kangaroo acetogens. Isolate TWA4 was a potent hydrogenotroph and demonstrated excellent mixotrophic growth (concomitant consumption of hydrogen during heterotrophic growth) with glycerol. Mixotrophic growth of isolate TWA4 on glycerol resulted in increased cell densities and acetate production compared to autotrophic growth. Co-cultures with an autotrophic methanogen Methanobrevibacter smithii revealed that isolate TWA4 performed reductive acetogenesis under high hydrogen concentration (> 5 mM), but not at low concentrations. Under heterotrophic growth conditions, isolate TWA4 did not significantly stimulate methanogenesis in a co-culture with M. smithii contrary to the expectation for organisms growing fermentatively.Conclusions: The unique properties of tammar wallaby acetogens might be contributing factors to reduced methanogen numbers and methane emissions from tammar wallaby forestomach fermentation, compared to ruminal fermentation. The macropod forestomach may be a useful source of acetogens for future strategies to reduce methane emissions from ruminants, particularly if these strategies also include some level of methane suppression and/or acetogen stimulation, for example by harnessing mixotrophic growth capabilities

An in vitro triple cell co-culture model with primary cells mimicking the human alveolar epithelial barrier

A triple cell co-culture model was recently established by the authors, consisting of either A549 or 16HBE14o- epithelial cells, human blood monocyte-derived macrophages and dendritic cells, which offers the possibility to study the interaction of xenobiotics with those cells. The 16HBE14o- containing co-culture model mimics the airway epithelial barrier, whereas the A549 co-cultures mimic the alveolar type II-like epithelial barrier. The goal of the present work was to establish a new triple cell co-culture model composed of primary alveolar type I-like cells isolated from human lung biopsies (hAEpC) representing a more realistic alveolar epithelial barrier wall, since type I epithelial cells cover >93% of the alveolar surface. Monocultures of A549 and 16HBE14o- were morphologically and functionally compared with the hAEpC using laser scanning microscopy, as well as transmission electron microscopy, and by determining the epithelial integrity. The triple cell co-cultures were characterized using the same methods. It could be shown that the epithelial integrity of hAEpC (mean ± SD, 1180 ± 188 Ω cm(2)) was higher than in A549 (172 ± 59 Ω cm(2)) but similar to 16HBE14o- cells (1469 ± 156 Ω cm(2)). The triple cell co-culture model with hAEpC (1113 ± 30 Ω cm(2)) showed the highest integrity compared to the ones with A549 (93 ± 14 Ω cm(2)) and 16HBE14o- (558 ± 267 Ω cm(2)). The tight junction protein zonula occludens-1 in hAEpC and 16HBE14o- were more regularly expressed but not in A549. The epithelial alveolar model with hAEpC combined with two immune cells (i.e. macrophages and dendritic cells) will offer a novel and more realistic cell co-culture system to study possible cell interactions of inhaled xenobiotics and their toxic potential on the human alveolar type I epithelial wall.

Sustained insulin secretory response in human islets co-cultured with pancreatic duct-derived epithelial cells within a rotational cell culture system

Aims/hypothesis - Loss of the trophic support provided by surrounding non-endocrine pancreatic cell populations underlies the decline in beta cell mass and insulin secretory function observed in human islets following isolation and culture. This study sought to determine whether restoration of regulatory influences mediated by ductal epithelial cells promotes sustained beta cell function in vitro. Methods - Human islets were isolated according to existing protocols. Ductal epithelial cells were harvested from the exocrine tissue remaining after islet isolation, expanded in monolayer culture and characterised using fluorescence immunocytochemistry. The two cell types were co-cultured under conventional static culture conditions or within a rotational cell culture system. The effect of co-culture on islet structural integrity, beta cell mass and insulin secretory capacity was observed for 10 days following isolation. Results - Human islets maintained under conventional culture conditions exhibited a characteristic loss in structural integrity and functional viability as indicated by a diminution of glucose responsiveness. By contrast, co-culture of islets with ductal epithelial cells led to preserved islet morphology and sustained beta cell function, most evident in co-cultures held within the rotational cell culture system, which showed a significantly (p<0.05) greater insulin secretory response to elevated glucose compared with control islets. Similarly, insulin/protein ratio data suggested that the presence of ductal epithelial cells is beneficial for the maintenance of beta cell mass. Conclusions/interpretation - The data indicate a supportive role for ductal epithelial cells in islet viability. Further characterisation of the regulatory influences may lead to novel strategies to improve long-term beta cell function both in vitro and following islet transplantation.

Aggravation of ADAMTS and MMP production and ERK1/2 pathway in the interaction of osteoarthritic subchondral bone osteoblasts and articular cartilage chondrocytes : a possible pathogenic role in osteoarthritis

Introduction: Degradative enzymes, such as A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) and matrix metalloproteinases (MMPs), play key roles in osteoarthritis (OA) development. The aim of the present study was to investigate if cross-talk between subchondral bone osteoblasts (SBOs) and articular cartilage chondrocytes (ACCs) in OA alters the expression and regulation of ADAMTS5, ADAMTS4, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9 and MMP-13, and also to test the possible involvement of mitogen activated protein kinase (MAPK) signaling pathway during this process. Methods: ACCs and SBOs were isolated from normal and OA patients. An in vitro co-culture model was developed to study the regulation of ADAMTS and MMPs under normal and OA joint cross-talk conditions. MAPK-ERK inhibitor, PD98059 was applied to delineate the involvement of specific pathway during this interaction process. Results: Indirect co-culture of OA SBOs with normal ACCs resulted in significantly increased expression of ADAMTS5, ADAMTS4, MMP-2, MMP-3 and MMP-9 in ACCs, whereas co-culture of OA ACCs led to increased MMP-1 and MMP-2 expression in normal SBOs. The upregulation of ADAMTS and MMPs under these conditions was correlated with activation of the MAPK-ERK1/2 signaling pathway and the addition of the MAPK-ERK inhibitor, PD98059, reversed the overexpression of ADAMTS and MMPs in co-cultures. Conclusion: In summary, we believe, these results add to the evidence that in human OA, altered bi-directional signals transmitted between SBOs and ACCs significantly impacts the critical features of both cartilage and bone by producing abnormal levels of ADAMTS and MMPs. Furthermore, we have demonstrated for the first time that this altered cross-talk was mediated by the phosphorylation of MAPK-ERK1/2 signaling pathway.

The co-transcriptome of uropathogenic Escherichia coli-infected mouse macrophages reveals new insights into host-pathogen interactions

Urinary tract infections (UTI) are among the most common infections in humans. Uropathogenic Escherichia coli (UPEC) can invade and replicate within bladder epithelial cells, and some UPEC strains can also survive within macrophages. To understand the UPEC transcriptional program associated with intramacrophage survival, we performed host–pathogen co-transcriptome analyses using RNA sequencing. Mouse bone marrow-derived macrophages (BMMs) were challenged over a 24 h time course with two UPEC reference strains that possess contrasting intramacrophage phenotypes: UTI89, which survives in BMMs, and 83972, which is killed by BMMs. Neither of these strains caused significant BMM cell death at the low multiplicity of infection that was used in this study. We developed an effective computational framework that simultaneously separated, annotated, and quantified the mammalian and bacterial transcriptomes. BMMs responded to the two UPEC strains with a broadly similar gene expression program. In contrast, the transcriptional responses of the UPEC strains diverged markedly from each other. We identified UTI89 genes upregulated at 24 h post-infection, and hypothesized that some may contribute to intramacrophage survival. Indeed, we showed that deletion of one such gene (pspA) significantly reduced UTI89 survival within BMMs. Our study provides a technological framework for simultaneously capturing global changes at the transcriptional level in co-cultures, and has generated new insights into the mechanisms that UPEC use to persist within the intramacrophage environment.

Time and Cell Type Dependency of Survival Responses in Co-cultured Tumor and Fibroblast Cells after Exposure to Modulated Radiation Fields

Advanced radiotherapy techniques such as intensity-modulated radiation therapy (IMRT) achieve high levels of conformity to the target volume through the sequential delivery of highly spatially and temporally modulated radiation fields, which have been shown to impact radiobiological response. This study aimed to characterize the time and cell type dependency of survival responses to modulated fields using single cell type (SCT) and mixed cell type (MCT) co-culture models of transformed fibroblast (AG0-1522b) cells, and prostate (DU-145) and lung (H460) cancer cells. In SCT cultures, in-field responses showed no significant time dependency while out-of-field responses occurred early, and plateaued 6 h after irradiation in both DU-145 and H460 cells. Under modulated beam configurations MCT co-cultures showed cell-specific, differential out-of-field responses depending on the irradiated in-field and responding out-of-field cell type. The observed differential out-of-field responses may be due to the genetic background of the cells, in particular p53 status, which has been shown to mediate radiation-induced bystander effects (RIBEs). These data provide further insight into the radiobiological parameters that influence out-of-field responses, which have potential implications for advanced radiotherapy modalities and may provide opportunities for biophysical optimization in radiotherapy treatment planning.

Effects of antihypertensive drugs in the differentiation and activation of human bone cells

O osso é um tecido metabolicamente ativo e a sua remodelação é importante para regular e manter a massa óssea. Esse processo envolve a reabsorção do material ósseo por ação dos osteoclastos e a síntese de novo material ósseo mediado pelos osteoblastos. Vários estudos têm sugerido que a pressão arterial elevada está associada a alterações no metabolismo do cálcio, o que leva ao aumento da perda de cálcio e da remoção de cálcio do osso. Embora as alterações no metabolismo ósseo sejam um efeito adverso associado a alguns fármacos antihipertensores, o conhecimento em relação a este efeito terapêutico ligado com os bloqueadores de canais de cálcio é ainda muito escasso. Uma vez que os possíveis efeitos no osso podem ser atribuídos à ação antihipertensiva dessas moléculas, ou através de um efeito direto nas atividades metabólicas ósseas, torna-se necessário esclarecer este assunto. Devido ao facto de que as alterações no metabolismo ósseo são um efeito adverso associado a alguns fármacos antihipertensores, o objetivo deste trabalho é avaliar o efeito que os bloqueadores dos canais de cálcio exercem sobre as células ósseas humanas, nomeadamente osteoclastos, osteoblastos e co-culturas de ambos os tipos celulares. Verificou-se que os efeitos dos fármacos antihipertensores variaram consoante o fármaco testado e o sistema de cultura usado. Alguns fármacos revelaram a capacidade de estimular a osteoclastogénese e a osteoblastogénese em concentrações baixas. Independentemente da identidade do fármaco, concentrações elevadas revelaram ser prejudiciais para a resposta das células ósseas. Os mecanismos intracelulares através dos quais os efeitos foram exercidos foram igualmente afetados de forma diferencial pelos diferentes fármacos. Em resumo, este trabalho demonstrou que os bloqueadores dos canais de cálcio utilizados possuem a capacidade de afetar direta- e indiretamente a resposta de células ósseas humanas, cultivadas isoladamente ou co-cultivadas. Este tipo de informação é crucial para compreender e prevenir os potenciais efeitos destes fármacos no tecido ósseo, e também para adequar e eventualmente melhorar a terapêutica antihipertensora de cada paciente.