169,981 research outputs found
Adjuvance of influenza virosomes in CTL induction in vitro
No full textThe induction of cytotoxic T lymphocyte (CTL) responses is of high relevance in immunological defense against intracellular pathogens and tumor cells. While humoral immune responses are successfully induced by a number of vaccines, the activation of cellular immune responses has only been addressed more recently. The development of novel immunogens from live attenuated vaccines to subunit vaccines demands efficient and safe adjuvants to improve their immunogenicity. Importantly, there are only three adjuvants licensed for human use: aluminium salts, MF59 (microfluidized detergent stabilized oil in water emulsion) and IRIV (immunopotentiating reconstituted influenza virosomes). Aluminium salts are the most widely used adjuvants and their efficacy in enhancement of humoral responses is well documented. They are ineffective in the induction of cellular responses, whereas IRIV and MF59 might be effective, in addition to humoral responses, also in the induction of cellular responses. The aim of our group, working in the field of cancer immunotherapy, is induction of CTL specific to melanoma associated antigens. The monitoring of a clinical phase I/II trial has demonstrated increased frequencies of specific CTL in peripheral blood upon administration of antigenic epitopes encoded as minigenes with costimulatory molecules in a recombinant vaccinia virus. In the heterologous vaccination protocol adopted, however, high CTL frequencies were not sustained upon administration of the same epitopes as synthetic peptides. This pattern prompted the search for appropriate adjuvants enhancing peptide induced CTL responses. In this thesis work we focused on the in vitro characterization of immune responses elicited by influenza virosomes and on the in vitro evaluation of influenza virosome adjuvance in HLA class I restricted peptide induced CTL responses. We tested empty IRIV admixed with peptides and influenza virosomes encapsulating peptides, both produced by Pevion Biotech Ltd. Due to the low encapsulation efficiency of IRIV per se, the production of the second formulation required encapsulation of peptides into liposomes and subsequent fusion with chimeric IRIV. Thus, we characterised immune responses elicited by empty IRIV and empty chimeric IRIV fused with empty liposomes (FCIRIV). Then, we evaluated their adjuvant capacity by testing CTL induction in the presence of IRIV admixed with peptides and by peptides encapsulated in FCIRIV as compared to CTL induction by peptides in absence of influenza virosomes. For IRIV admixed with peptides we addressed induction of CTL specific for the highly immunogenic Influenza matrix 58-66 (IM58-66) and to the immunodominant melanoma
associated Melan-A/ Mart-127-35 HLA-A201 restricted epitopes. For peptides encapsulated
in FCIRIV we addressed induction of CTL specific for the L27Melan-A/Mart-126-35 HLAA0201
restricted epitope.
Our results demonstrate that all influenza virosome formulations under investigation
induce antigen triggered CD4+ T cell proliferation characterized by a T helper 1 cytokine
profile. Further dissection of CD4+ T cells identified CD4+CD45RO+ cells as proliferative
responders to IRIV stimulation and no major cell proliferation could be induced in cord
blood mononuclear cell cultures. These findings indicate that the majority of CD4+ T cells
responding to IRIV are antigen experienced. In addition, supernatants of IRIV stimulated
PBMC cultures favoured maturation of dendritic cells, as demonstrated by upregulation of
HLA-ABC, CD86 and CD83.
Both, influenza virosomes admixed with peptides or encapsulating peptides significantly
enhanced specific CTL induction, as detected by multimer staining and cytotoxicity assays.
CTL induction experiments in presence of irradiated CD4+ T cells indicated that IRIV CTL
adjuvance required CD4+ T cell activation. In addition, transwell cultures pointed to a key
role of cytokines in IRIV mediated CTL adjuvance.
In contrast to empty IRIV, FCIRIV with encapsulated peptides were characterized by CD4+
T cell independent adjuvant potential, possibly attributable to influenza virosome delivery
capacities.
Taken together, our results demonstrate that influenza virosomes are endowed with the
capacity to enhance HLA class I restricted CTL induction in vitro. Importantly, this could be
demonstrated not only for the highly immunogenic IM58-66 epitope, but also for the
melanoma associated epitopes L27Melan-A/Mart-126-35 and Melan-A/Mart-127-35.
Moreover, CTL induced by L27Melan-A/Mart-126-35 encapsulated in FCIRIV were capable of
recognizing and lysing tumor cells that constitutively express the Melan-A/Mart-1 antigen.
These in vitro findings encourage further evaluation of influenza virosome CTL adjuvance
in vivo
Indoor and HVAC air quality investigation, China Basin - Source characterization, risk assessment and health effects
No full textVEGF-expressing mesenchymal stem cells for improved angiogenesis in regenerative medicine : a bone tissue engineering approach
No full textRapid vascularization of tissue-engineered grafts is a major bottleneck in the development of regenerative medicine approaches. In order to overcome this limitation, we aimed to develop a bone tissue engineering strategy combining cell therapy with pro-angiogenic gene therapy.
Vascular Endothelial Growth Factor (VEGF) is the master regulator of physiological vascular growth and is commonly used as a therapeutic transgene for the induction of angiogenesis. However, uncontrolled and high levels of VEGF expression can lead to aberrant vascular growth. To achieve controlled expression in vivo, a high-throughput flow cytometry-based method has previously been developed in our group. Linking the VEGF cDNA to a cell-surface marker (a truncated version of CD8a) in a bicistronic construct enabled the rapid purification of genetically modified myoblasts secreting a desired VEGF level, using FACS sorting based on the intensity of CD8 expression in each cell. Controlled VEGF expression in skeletal muscle, achieved by implantation of these FACS-purified myoblast populations, induced only normal, stable and functional vascular networks and avoided any aberrant angiogenesis.
The aims of this thesis were to adapt this method to human adipose tissue- and bone marrow-derived mesenchymal stromal/stem cells (ASC and BMSC), and to apply these in a bone tissue engineering approach to increase the vascularization potential of osteogenic grafts.
As MSC gradually loose their regenerative potential during in vitro expansion, we first optimized our genetic engineering method for MSC, so as to enable high transduction efficiency and FACS-purification with minimal in vitro manipulation. Chapter 2 describes the generation of an optimized protocol allowing routine transduction efficiencies of > 90% of primary human ASC and BMSC already during the first plating, as well as flow cytometry purification of transduced cells at the time of the first passage. In addition we demonstrated that it was possible to FACS-purify specific sub-populations of transduced MSC homogeneously producing desired VEGF doses. Neither retroviral vector transduction, FACS-purification, nor the expression of the transgenes VEGF and CD8 impaired MSC proliferation and in vitro differentiation potential. Transgene expression was not lost during in vitro differentiation.
In Chapter 3, proof-of-principle was obtained by applying this platform to a bone tissue engineering approach. Human BMSC, transduced and rapidly FACS-purified to eliminate non-expressing cells, were seeded onto hydroxyapatite granules to generate non-critically sized constructs, and were implanted subcutaneously in nude rats. In vivo vascularization potential was significantly increased in VEGF-expressing BMSC. Although VEGF expression was heterogeneous, no aberrant angiogenesis was observed. Indeed, orderly vascular beds were induced, with flow-conducting arterioles feeding into extensive capillary networks, where metabolic exchanges can take place efficiently. The improvement in vascularization was not diminished by extensive in vitro expansion of the transduced BMSC up to 35 population doublings, showing that genetic modification conferred a stable angiogenic potential. As expected, these expanded BMSC lost their osteogenic potential. However, their sustained capacity to induce vascularization could be useful in other applications, where effective expansion of the vascular bed is required, but not progenitor differentiation, such as in cell-based approaches for therapeutic angiogenesis in peripheral or coronary artery diseases.
By minimizing cell expansion, both naïve and control transduced MSC generated abundant bone tissue in vivo. However, VEGF over-expression specifically caused a strong reduction in bone formation. This correlated with an increased recruitment of TRAP-positive osteoclasts specifically in VEGF-expressing constructs.
These data suggest that VEGF over-expression might impair bone formation by disrupting the balance between bone formation and resorption towards excessive degradation. To fully understand the underlying mechanism, further experiments will be needed.
The method described in chapter 2 provides a general platform to generate populations of genetically modified MSC, expressing specific levels of a therapeutic transgene, already at the time of the first passage. Therefore, it has the potential to be applied in other fields of regenerative medicine, beyond bone tissue engineering. We briefly describe two recently initiated projects, based on the results described in this thesis, which aim at either promoting or inhibiting angiogenesis in order to improve cardiac function after myocardial infarction, or cartilage tissue formation, respectively
Engineering of cartilage tissue constructs in a 3-dimensional perfusion bioreactor culture system under controlled oxygen tension
No full textThe most relevant results generated in this thesis can be summarized as follow:
· Adult human articular chondrocytes (AHAC) from elderly individuals expanded in culture
medium supplemented with the growth factors TGFβ-1, FGF-2 and PDGF and subsequently
cultured in 3-d pellets had an enhanced chondrogenic capacity when exposed to more
physiological (i.e. 5%) oxygen levels.
· In correlation with the enhanced tissue forming capacity of AHAC from elderly donors under
low oxygen tension, the mRNA expression levels of selective matrix degrading enzymes
were reduced as compared to conventional in vitro oxygen culture condition.
· We developed an integrated bioreactor system, which streamlines within a single device the
phases of perfusion cell seeding and prolonged perfusion culture of cell seeded scaffolds in
vitro.
· The culturing of uniformly seeded adult human articular chondrocytes under direct perfusion,
where cells are continuously exposed to a normoxic range of oxygen levels, can maintain a
uniform distribution of viable cells throughout thick porous scaffolds as compared to
statically cultured constructs.
· The culturing of constructs uniformly seeded with adult human articular chondrocytes under
a more physiological range of oxygen resulted in a higher chondrogenic differentiation as
compared to culture under normoxic levels. Anyhow, this effect was less pronounced as
compared to statically cultured cell constructs or micromass cell pellets, possibly due to the
flow induced shear forces.
· Reduced perfusion flow rates applied to chondrocytes on porous scaffolds significantly
induced more cartilaginous tissue in the presents of low vs. high oxygen levels. However the
effects of low oxygen were not as marked as in pellet culture
Going Beyond Counting First Authors in Author Co-citation Analysis
No full textThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Mitomycin C in highly myopic eyes - Author reply
No full textOphthalmology. 2005 Feb;112(2):208-18; discussion 219.
Mitomycin C modulation of corneal wound healing after photorefractive keratectomy in highly myopic eyes.
Gambato C, Ghirlando A, Moretto E, Busato F, Midena E.
SourceRefractive Surgery Service and Antimetabolite Therapy Research Unit, Department of Ophthalmology, University of Padova, Padova, Italy.
Abstract
PURPOSE: To evaluate the role of topical mitomycin C in corneal wound healing (CWH) after photorefractive keratectomy (PRK) in highly myopic eyes.
DESIGN: Prospective, double-masked, randomized clinical trial.
PARTICIPANTS: Seventy-two eyes of 36 patients affected by high (>7 diopters) myopia.
METHODS: In each patient, one eye was randomly assigned to PRK with intraoperative topical 0.02% mitomycin C application, and the fellow eye was treated with a placebo. Postoperatively, mitomycin C-treated eyes received artificial tears (3 times daily, tapered in 3 months), whereas the fellow eye was treated with fluorometholone sodium 2% and artificial tears (3 times daily, tapered in 3 months).
MAIN OUTCOME MEASURES: Uncorrected visual acuity (UCVA) and best-corrected visual acuity (BCVA), contrast sensitivity, manifest refraction, and biomicroscopy. Contrast sensitivity was determined using the Pelli-Robson chart. Corneal confocal microscopy documented CWH.
RESULTS: Mean follow-up was 18 months (range, 12-36). No side effects or toxic effects were documented. At 12-month follow-up examination, UCVAs (logarithm of the minimum angle of resolution) were 0.4+/-0.48 and 0.5+/-0.53 (P = .03) in mitomycin C-treated eyes and corticosteroid-treated eyes, respectively. At 1 year, corneal haze developed in 20% of corticosteroid-treated eyes, versus 0% of mitomycin C-treated eyes. At 12, 24, and 36 months, corneal confocal microscopy showed activated keratocytes and extracellular matrix significantly more evident in untreated eyes (Ps = 0.004, 0.024, and 0.046, respectively).
CONCLUSION: Topical intraoperative application of 0.02% mitomycin C can reduce haze formation in highly myopic eyes undergoing PRK.
Comment in
Ophthalmology. 2006 Feb;113(2):357; author reply 357-8
China, the European Union and the United States of America: Partners or competitors?
No full textThis working paper brings together assessments of the trilateral relations between China, the EU und the US from the disciplines of political science and economics. The first paper primarily addresses the following three issues: a) China's current development and prospects b) EU-China relations, and c) recommendations in terms of EU policies towards China. The second paper applies a three tier analysis looking first at changes in the Chinese and global economic setting caused by China's WTO accession. It then takes a look at the bilateral economic relations between China and the EU and US respectively. Finally the scored goals and competing interests of the USA and the EU vis-à-vis China are analysed. --EU-US-China relations,China's domestic development,EU policies,China's external economic relations,China's WTO accession,structural change,World economy
Generation of osteoinductive grafts by three-dimensional perfusion culture of human bone marrow cells into porous ceramic scaffolds
No full textThe main aims of this thesis were (i) to identify and develop a system that could be
reproducibly used to streamline manufacture of osteoinductive grafts based on human bone marrow
stromal cells (BMSC) in the context of regenerative medicine, (ii) to characterize the developed
system in order to identify key elements responsible for its reproducible and efficient performance,
and (iii) to extend its use to a sheep cell source, thus opening the way to test the osteoinductivity of
orthotopic implants in a large animal model.
Bone Marrow Stromal Cells (BMSC), which are typically defined by their capacity to adhere
on plastic [1] and form a fibroblastic colony (CFU-f) [2], represent a very low fraction (approximately
0.01%) among the nucleated cells of the bone marrow. Therefore, to obtain a sufficient number of
cells for bone tissue engineering applications, BMSC are typically first selected and expanded in
monolayer (2D) prior to loading into 3D scaffolds. However, 2D-expansion causes BMSC to
progressively lose their early progenitor properties and differentiation potential [3-5], and to decrease
their capability to form colonies and to induce bone tissue formation upon ectopic implantation [3],
placing several potential limits on their clinical utility. To bypass the process of 2D-expansion and its
associated limitations, we used an innovative bioreactor-based approach to seed, expand, and
differentiate BMSC directly in a 3D ceramic scaffold [6]. Nucleated cells, freshly isolated from a bone
marrow aspirate, were introduced into the bioreactor system and perfused through the pores of 3D
ceramics for five days, then further cultured under perfusion for an additional two weeks. Using the
developed procedure, BMSC could be seeded and extensively expanded within the 3D environment of
the ceramic pores. Interestingly, we found that the 3D-generated constructs contained both
hemopoietic cells and BMSC, whose relative fractions could be modulated by appropriate media
supplements, and that a consistent fraction of expanded BMSC was clonogenic. In contrast, following
the typical 2D-expansion, cells of the hemopoietic lineage could not be maintained, and, consistently
with previous studies, only a minor fraction of expanded BMSC was still clonogenic. When constructs
were ectopically implanted in nude mice, those engineered in the bioreactor reproducibly generated
bone tissue that was uniformly distributed throughout the scaffold volume and filled up to 60% of the
ceramic pores. In marked contrast, when similar numbers of 2D-expanded BMSC were loaded into
ceramic scaffolds and implanted, bone was infrequently generated, and even in the most
osteoinductive constructs, it was localized to peripheral regions, filling only 10% of the ceramic pore
volume [6].
Considering the need of reproducibility or at least of predictability in the osteoinductive ability
of the constructs for their standardized clinical use, in order to validate the possibility of extending the
use of the developed bioreactor-based approach for generating osteoinductive grafts of clinically
relevant size, we then investigated whether a minimum cell density was required for the reproducible
bone tissue formation. Based on the established association between the higher clonogenicity of
BMSC expanded in the 3D-system and the more reproducible and extensive osteoinductivity of the
resulting constructs, as compared to those based on 2D-expanded BMSC, we demonstrated that
presence or absence of bone in the constructs following ectopic implantation is related not to the total
number of implanted BMSC, but to the number of CFU-f present in the construct at the time of
implantation. In particular, we identified an apparent threshold in the amount of CFU-f discriminating
between osteoinductive and not osteoinductive constructs.
The developed bioreactor-based approach has been validated in a heterotopic model. Before
envisioning a clinical trial in human, a study in a large animal model is needed to validate the safety
and the surgical feasibility of the overall procedure. Thus, in the perspective of testing our novel
approach for repairing experimental bone defects in a sheep model, it was first necessary to validate
our system using ovine BMSC. We demonstrated that osteoinductive constructs can be generated by
perfusing 3D ceramic scaffolds with the nucleated cell fraction of ovine bone marrow aspirates [7].
Ongoing studies in the context of an EU-funded Project are aimed at testing the capability of the
generated constructs to repair large bone defects in sheep (i.e. defects around titanium implants
inserted into trabecular bone of the proximal humerus, and postero-lateral spinal fusion in lumbarregion)
Dispelling the Myths Behind First-author Citation Counts
No full textWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
A Multi-Language Comparison of Influences on Author Verification using Character N-Grams
No full textWe create a new multi-language corpus for author verification based on Wikipedia talkpages, and evaluate the influence that differences in topic and time have on character n-gram author profiles. Topic alignment between two texts is found to increase author verification precision, and an authors writing style is found to change over time, but not more significantly after 3 years than after 1 year.Information ArchitectureWISElectrical Engineering, Mathematics and Computer Scienc
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