Engraftment and tolerance induction in cellular therapy : focus on mesenchymal stromal cells

Different cell types have been employed in the search for a cellular therapy that is applicable for the treatment of heart failure. Remuscularization strategies using stem-cell-derived cardiomyocytes have been prone to arrhythmias. Mesenchymal stromal cells (MSCs) are one of the most promising cell types for the treatment of heart failure due to their diverse regenerative properties. We have isolated MSCs from the human fetal heart in the form of human fetal cardiac MSCs (hfcMSCs), which might comprise a more relevant cell source than bone marrow-derived MSCs, for the treatment of heart failure. Although the cell source is of importance for successful cell therapy, the efficacy of the cells also depends on avoiding anchorage-dependent apoptosis (anoikis) and immune rejection. Immune rejection may be counteracted with immunosuppressive drugs; however, these drugs generally have serious side effects that limit their suitability in cell therapy. We have used a transient blockade of T cell costimulation with the aim to induce immunologic tolerance through the generation of Regulatory T cells (Tregs) specifically for the antigens of the transplant. Anoikis may be diminished by the simultaneous administration of the exogenous extracellular matrix (ECM), which provides anchorage possibilities for the transplanted cells. In Paper I and II the use of costimulation blockade in xenogeneic and allogeneic mouse models was explored and we showed that Foxp3+ Tregs are associated with tolerated and viable grafts as well as reduced reactivity in mixed lymphocyte reaction tests. MSCs, known for their immunomodulatory properties, were shown to synergize with costimulation blockade to facilitate immunological tolerance to allogeneic insulin-producing islets in mice. In Paper III we showed that Matrigel synergizes with costimulation blockade to enable enhanced cell retention of hfcMSCs after subcutaneous implantation in mice. The hfcMSCs displayed vasculogenic potential in vivo, shown as the expression of CD31, and also the formation of vessel-like structures staining positive for laminin a4. In Paper IV, hfcMSCs were cultured on a 3D scaffold composed of a novel type of artificial spider silk, NT2RepCT, to conserve the cell connections and ECM at the time of implantation. The hfcMSCs displayed vasculogenic potential in the 3D cultures on NT2RepCT as well, with the formation of an ECM that stained positive for laminin a4 and fibronectin and contained vessel-like structures expressing CD31. After in vivo implantation, the hfcMSCs on the NT2RepCT were subjected to immune rejection regardless of costimulation blockade or isotype control treatment. In this thesis hfcMSCs were shown to have a vasculogenic potential as they also deposit laminins that are important for vascular structures and thereby may have the potential to prevent ischemic heart failure. We also demonstrated a synergistic effect between costimulation blockade and MSCs or Matrigel for enhanced cell retention. Artificial spider silk showed great potential for advanced in vitro studies with 3D cultures, but could not be used in vivo in its current form

Heavy metal discharge into Lake Victoria : a study of the Ugandan cities of Kampala, Jinja and Entebbe

Water samples from different sampling sites located in the Ugandan cities of Kampala, Jinja and Entebbe as well as the remote Ssese Islands, were analyzed for concentrations of Cd, Cr, Cu, Ni, Pb and Zn. Both Kampala and Jinja have large industrial areas while Entebbe has Uganda's largest airport. Household and industrial wastes are discharged into Lake Victoria largely untreated. The objectives of this study were to: 1) evaluate concentrations of the metals in discharge from the selected sites; 2) determine whether or not wetlands impacted on the magnitude of metal discharge into the lake, 3) determine and assess heavy metal levels in drinking and irrigation water; 4) evaluate heavy metal related risks for humans and the environment. The discharges from the cities contained rather high concentrations of Cd, Cu, Pb and Zn in comparison with Swedish environmental guidelines. In Kampala and Jinja, samples were taken before and after water had passed through areas of wetland. The results show a reduction of these metals as the water passed through these areas. The reduction was especially noticeable in Kampala, where metal concentrations were reduced by up to 99%. The concentrations of all metals but Ni were alarming in Lugogo and Mpanga along Nakivubo channel (running through central and industrial areas in Kampala), these sites also being used for household farming. Some of the results from Jinja could be explained by the former Cu mine at Kilembe. The activity at the airport of Entebbe seemed to cause heavy metal pollution in nearby waters. Areas of special concern regarding the environment were all sites along Nakivubo channel, especially since the concentrations of Cd, Pb and Zn were high already at its emergence point in Makerere. In Jinja and Entebbe, all sites had Cd- and Zn concentrations exceeding the given guideline for sub-lethal or chronic effects. The expected result of lower concentrations in water outside Kalangala of Ssese Islands than outside Kampala, Jinja and Entebbe was not confirmed. The finding that the Ssese Islands had higher concentrations of Pb and Cr than 55% of the sampling sites in the cities was most surprising. Only 45% of the city sites had lower Cu concentrations than the Ssese Islands. The high concentration of Pb at Ssese Islands is alarming. The concentration was above the Swedish environmental guideline used, and there were no point sources of this metal on the island. Possible sources are therefore transport via air and/or water from the riparian countries as well as unauthorized dumping of wastes. The results from all sampling sites were compared with WHO's guidelines for drinking water. Only the Kampala sites Makerere, Lugogo and Mpanga exceeded the guidelines for Pb. Similarly, Cd and Ni concentrations at Makerere and Lugogo exceeded the guidelines. Concentrations of Pb and Cd were high at the majority of the sites. This is alarming since these metals are among the most hazardous to humans and the environment. Reduced discharge of the other metals is also important since low concentrations can accumulate and have negative longterm effects. The importance of wetlands as a purifying media must be recognized and protected. Efforts should also be put into limiting the sources of metals since treatment always means that it will end up somewhere else. Conversion to unleaded petrol as well as proper maintenance of dumping sites could lead to reduced metal leakage and decreased unlawful dumping in the lake. Implementation and enforcement of environmental legislation are most important

Breastfeeding does not influence the development of inhibitors in haemophilia.

Our aim was to test the hypothesis that breastfeeding may reduce development of inhibitors in male infants with haemophilia by inducing an oral immune tolerance to factor VIII. To achieve that goal, we performed a structured epidemiological survey comprising all males born with severe haemophilia A (in all 100 patients, 19 with inhibitors) or haemophilia B (in all 16 patients, six with inhibitors) in Sweden in 1980-99. Our results show no protective effect of breastfeeding

Broadening the roles of UDP-glycosyltransferases in auxin homeostasis and plant development

The levels of the important plant growth regulator indole-3-acetic acid (IAA) are tightly controlled within plant tissues to spatiotemporally orchestrate concentration gradients that drive plant growth and development. Metabolic inactivation of bioactive IAA is known to participate in the modulation of IAA maxima and minima. IAA can be irreversibly inactivated by oxidation and conjugation to aspartate and glutamate. Usually overlooked because of its reversible nature, the most abundant inactive IAA form is the IAA-glucose (IAA-glc) conjugate. Glycosylation of IAA in Arabidopsis thaliana is reported to be carried out by UDP-glycosyltransferase 84B1 (UGT84B1), while UGT74D1 has been implicated in the glycosylation of the irreversibly formed IAA catabolite oxIAA. Here we demonstrated that both UGT84B1 and UGT74D1 modulate IAA levels throughout plant development by dual IAA and oxIAA glycosylation. Moreover, we identified a novel UGT subfamily whose members redundantly mediate the glycosylation of oxIAA and modulate skotomorphogenic growth

PIF7 is a master regulator of thermomorphogenesis in shade

Plant hypocotyl elongation response to light and temperature. Here the authors show that shade combined with warm temperature synergistically enhances the hypocotyl growth response via the PIF7 transcription factor, auxin, and as yet unknown factor.The size of plant organs is highly responsive to environmental conditions. The plant's embryonic stem, or hypocotyl, displays phenotypic plasticity, in response to light and temperature. The hypocotyl of shade avoiding species elongates to outcompete neighboring plants and secure access to sunlight. Similar elongation occurs in high temperature. However, it is poorly understood how environmental light and temperature cues interact to effect plant growth. We found that shade combined with warm temperature produces a synergistic hypocotyl growth response that dependent on PHYTOCHROME-INTERACTING FACTOR 7 (PIF7) and auxin. This unique but agriculturally relevant scenario was almost totally independent on PIF4 activity. We show that warm temperature is sufficient to promote PIF7 DNA binding but not transcriptional activation and we demonstrate that additional, unknown factor/s must be working downstream of the phyB-PIF-auxin module. Our findings will improve the predictions of how plants will respond to increased ambient temperatures when grown at high density

Vernalization shapes shoot architecture and ensures the maintenance of dormant buds in the perennial Arabis alpina

Perennials have a complex shoot architecture with axillary meristems organized in zones of differential bud activity and fate. This includes zones of buds maintained dormant for multiple seasons and used as reservoirs for potential growth in case of damage. The shoot of Arabis alpina, a perennial relative of Arabidopsis thaliana, consists of a zone of dormant buds placed between subapical vegetative and basal flowering branches. This shoot architecture is shaped after exposure to prolonged cold, required for flowering.To understand how vernalization ensures the maintenance of dormant buds, we performed physiological and transcriptome studies, followed the spatiotemporal changes of auxin, and generated transgenic plants.Our results demonstrate that the complex shoot architecture in A. alpina is shaped by its flowering behavior, specifically the initiation of inflorescences during cold treatment and rapid flowering after subsequent exposure to growth-promoting conditions. Dormant buds are already formed before cold treatment. However, dormancy in these buds is enhanced during, and stably maintained after, vernalization by a BRC1-dependent mechanism. Post-vernalization, stable maintenance of dormant buds is correlated with increased auxin response, transport, and endogenous indole-3-acetic acid levels in the stem.Here, we provide a functional link between flowering and the maintenance of dormant buds in perennials

The RPN12a proteasome subunit is essential for the multiple hormonal homeostasis controlling the progression of leaf senescence

The 26S proteasome is a conserved multi-subunit machinery in eukaryotes. It selectively degrades ubiquitinated proteins, which in turn provides an efficient molecular mechanism to regulate numerous cellular functions and developmental processes. Here, we studied a new loss-of-function allele of RPN12a, a plant ortholog of the yeast and human structural component of the 19S proteasome RPN12. Combining a set of biochemical and molecular approaches, we confirmed that a rpn12a knock-out had exacerbated 20S and impaired 26S activities. The altered proteasomal activity led to a pleiotropic phenotype affecting both the vegetative growth and reproductive phase of the plant, including a striking repression of leaf senescence associate cell-death. Further investigation demonstrated that RPN12a is involved in the regulation of several conjugates associated with the auxin, cytokinin, ethylene and jasmonic acid homeostasis. Such enhanced aptitude of plant cells for survival in rpn12a contrasts with reports on animals, where 26S proteasome mutants generally show an accelerated cell death phenotype.A loss-of-function mutation in the RPN12a proteasome subunit in Arabidopsis thaliana shows that this protein is essential for hormonal homeostasis, thereby modulating plant's development and the progression of leaf senescence

Fluorescence activated cell sorting-A selective tool for plant cell isolation and analysis

Instrumentation for flow cytometry and sorting is designed around the assumption that samples are single-cell suspensions. However, with few exceptions, higher plants comprise complex multicellular tissues and organs, in which the individual cells are held together by shared cell walls. Single-cell suspensions can be obtained through digestion of the cells walls and release of the so-called protoplasts (plants without their cell wall). Here we describe best practices for protoplast preparation, and for analysis through flow cytometry and cell sorting. Finally, the numerous downstream applications involving sorted protoplasts are discussed

Alleviation of Zn toxicity by low water availability

Heavy metal contamination and drought are expected to increase in large areas worldwide. However, their combined effect on plant performance has been scantly analyzed. This study examines the effect of Zn supply at different water availabilities on morpho-physiological traits of Quercus suber L. in order to analyze the combined effects of both stresses. Seedlings were treated with four levels of zinc from 3 to 150 µM and exposed to low watering (LW) or high watering (HW) frequency in hydroponic culture, using a growth chamber. Under both watering regimes, Zn concentration in leaves and roots increased with Zn increment in nutrient solution. Nevertheless, at the highest Zn doses, Zn tissue concentrations were almost twice in HW than in LW seedlings. Functional traits as leaf photosynthetic rate and root hydraulic conductivity, and morphological traits as root length and root biomass decreased significantly in response to Zn supply. Auxin levels increased with Zn concentrations, suggesting the involvement of this phytohormone in the seedling response to this element. LW seedlings exposed to 150 µM Zn showed higher root length and root biomass than HW seedlings exposed to the same Zn dose. Our results suggest that low water availability could mitigate Zn toxicity by limiting internal accumulation. Morphological traits involved in the response to both stresses probably contributed to this response.This research was funded by the Spanish Ministry of Science and Innovation (Project GRACCIE, Programa Consolider-Ingenio 2010 (CSD 2007-00067) and SURVIVE (CGL-2011-30531-CO2-02)) and Generalitat Valenciana (FEEDBACKS-PROMETEO/2009/006). E. I. Hernández thanks the University of Alicante for her FPU research fellowship. CEAM is supported by Generalitat Valenciana

Studies of moss reproductive development indicate that auxin biosynthesis in apical stem cells may constitute an ancestral function for focal growth control

The plant hormone auxin is a key factor for regulation of plant development, and this function was probably reinforced during the evolution of early land plants. We have extended the available toolbox to allow detailed studies of how auxin biosynthesis and responses are regulated in moss reproductive organs, their stem cells and gametes to better elucidate the function of auxin in the morphogenesis of early land plants. We measured auxin metabolites and identified IPyA (indole-3-pyruvic acid) as the main biosynthesis pathway inPhyscomitrium(Physcomitrella)patensand established knock-out, overexpressor and reporter lines for biosynthesis genes which were analyzed alongside previously reported auxin-sensing and transport reporters. Vegetative and reproductive apical stem cells synthesize auxin. Sustained stem cell activity depends on an inability to sense the auxin produced while progeny of the stem cells respond to the auxin, aiding in the control of cell division, expansion and differentiation. Gamete precursors are dependent on a certain degree of auxin sensing, while the final differentiation is a low auxin-sensing process. Tha data presented indicate that low auxin activity may represent a conserved hallmark of land plant gametes, and that local auxin biosynthesis in apical stem cells may be part of an ancestral mechanism to control focal growth