Limbal stem cell deficiency (LSCD) is a disease resulting from the loss or dysfunction of epithelial stem cells, which seriously impairs sight. Autologous limbal stem cell transplantation is ...effective in unilateral or partial bilateral disease but not applicable in total bilateral disease. An allogeneic source of transplantable cells for use in total bilateral disease can be obtained from culture of donated cadaveric corneal tissue. We performed a controlled multicenter study to examine the feasibility, safety, and efficacy of allogeneic corneal epithelial stem cells in the treatment of bilateral LSCD. Patients were randomized to receive corneal epithelial stem cells cultured on amniotic membrane (AM): investigational medicinal product (IMP) or control AM only. Patients received systemic immunosuppression. Primary endpoints were safety and visual acuity, secondary endpoint was change in composite ocular surface score (OSS). Sixteen patients were treated and 13 patients completed all assessments. Safety was demonstrated and 9/13 patients had improved visual acuity scores at the end of the trial, with no significant differences between IMP and control groups. Patients in the IMP arm demonstrated significant, sustained improvement in OSS, whereas those in the control arm did not. Serum cytokine levels were measured during and after the period of immune suppression and we identified strongly elevated levels of CXCL8 in the serum of patients with aniridia, which persisted throughout the trial. This first randomized control trial of allogeneic corneal epithelial stem cells in severe bilateral LSCD demonstrates the feasibility and safety of this approach. Stem Cells Translational Medicine 2019;8:323–331
Patients with severe ocular surface disorder received transplants of amniotic membrane with (black bars) or without (gray bars) cadaveric‐donor‐derived cultured limbal stem cells. All patients received immune suppression. Only patients who received transplants containing limbal stem cells showed sustained significant improvements (reductions) in combined ocular surface scores (5 factors scored 0–3 where 0 is a normal eye score).
This thesis plots a practice-based Collaborative Doctoral Award working in partnership with York Art Gallery, the British Ceramics Biennial, Manchester School of Art and the Stoke-on-Trent ceramics ...manufacturer Cauldon Ceramics. The project was animated by a central question: 'How can holistic design practices of the individual designer-maker inform and enhance the design aesthetics and design practices of the industrial ceramic manufacturer?' This thesis is an investigation of the phenomenon of the individual designer-maker and their potential to act as a catalyst for industrial ceramic innovation. The centring of practice recognises that the maker's professional skills and expertise produce a specific valuable contribution to knowledge creation. I understand my role within this research as a hybrid 'designer-practitioner-researcher' (Vaughan, 2019). Cauldon Ceramics is the last remaining producer of the Brown Betty teapot - a traditional design that originated in Stoke-on-Trent in the mid-eighteenth century. Once produced in the millions per year, it has been in decline since the late 1970s. Through a process of primary archival research, literature reviews, site visits, material experimentation and prototyping, as well as public discourse in the form of exhibitions, talks and publications, I identified Cauldon Ceramics as an appropriate manufacturer to test a live case study. My research ascertained that the Brown Betty should be revitalised for a number of reasons: A Brown Betty made in Staffordshire has cultural significance; There is a lack of historical and contemporary understanding of the object and inconsistencies within the available literature; The design details of the product itself have deteriorated over the last 40 years indicating that the Brown Betty has both evolved and deteriorated; The cultural significance of the object is being lost in the design, manufacture and promotion of both the contemporary Staffordshire made versions and overseas imported versions. During this practice-based research I have re-discovered a forgotten innovative past, re-defined the cultural significance of the Brown Betty, identified historical precedents in the design and manufacturing of the object, developed new markets, and cultivated and galvanised stakeholders. I have re-engineered and re-launched the object through a process of re-design and the re-introduction of innovative historical patents, contemporary design details and new manufacturing processes. The result is a revitalised object named the 'Re-engineered Brown Betty' teapot.
Summary
Mannan‐binding lectin (MBL) is a collectin synthesized by the liver and secreted into the bloodstream. It has a receptor for microbial structures in its C‐type lectin domain and a separate ...receptor(s) located within its collagen‐like region for autologous phagocytic cells. Here we demonstrate that human peripheral blood adherent cells (monocytes) and monocyte‐derived dendritic cells are a source of MBL, and that a novel calcium‐dependent and sugar‐specific MBL receptor is up‐regulated in immature (CD1a‐positive) dendritic cells. These findings suggest a previously unsuspected autologous function for MBL, perhaps a regulatory role within the immune system.
Abstract Mannan (or mannose)–binding lectin (MBL) can bind to monocytes and dendritic cells, but the significance of such interactions is unknown. We hypothesized that the presence of MBL might ...prevent the differentiation of monocytes into monocyte-derived dendritic cells or interfere with the development of dendritic cells in some way. We therefore investigated the influence of recombinant human MBL on surface antigen expression and on secretion of selected cytokines. By these means, no direct influence of rhMBL on dendritic cell differentiation or maturation was detected. However, mature dendritic cells prepared in the presence of rhMBL and subsequently co-cultured with allogeneic mononuclear cells, markedly promoted production of interleukin-1β, interleukin-6, and tumor necrosis factor–α in vitro . In most dendritic cell–mononuclear cell combinations, IFN-γ production was also enhanced. This influence required the presence of rhMBL during dendritic cell maturation and was critically dependent on the presence of monocytes. This observation provides evidence that MBL can influence cellular immunity in addition to its established role as an opsonin.
MBL (mannan-binding lectin; also called mannose-binding lectin) is a circulating C-type lectin with a collagen-like region synthesized mainly by the liver. MBL may influence susceptibility to ...infection in recipients of stem cell transplants, and it has even been suggested that the MBL status of a donor can influence the recipient's susceptibility to post-transplant infections. We have previously reported that MBL can be detected on human monocytes and monocyte-derived dendritic cells, based on detection using biotinylated anti-MBL, suggesting that those cells could synthesize MBL. If true, permanent MBL replacement therapy could be achieved by stem cell infusions. However, two other groups independently failed to find mbl-2-derived mRNA in monocytes. Therefore, to confirm or refute our previous observations, we used an alternative experimental strategy. Instead of using biotinylated antibody and labelled streptavidin, detection of surface MBL was attempted using MBL-specific primary antibodies (131-1, 131-10 and 131-11) followed by fluorescein-labelled anti-IgG, and controlled by the use of non-specific IgG as primary antibody. Monocytes were counterstained with anti-CD14-PE before FACS analysis. Adherent monocytes were also cultured for 48 h in serum-free medium or converted into immature dendritic cells by culture with IL-4 (interleukin-4) and GM-CSF (granulocyte/monocyte colony-stimulating factor). During FACS analysis, the dendritic cells were gated after counter-staining with anti-CD1a-PE. MBL was readily detected on the surface of fresh monocytes using all three specific anti-MBL monoclonal antibodies, but specific anti-MBL binding was greatly diminished after monocytes had been cultured for 2 days in serum-free medium. Moreover, we could not detect any MBL present on the surface of monocyte-derived dendritic cells. We therefore conclude that MBL is indeed present on the surface of fresh human monocytes. However, in view of the mRNA findings of others and our own previous observation that no secretion of MBL took place in culture, we presume that the surface-bound MBL is derived from autologous plasma and not synthesized by the cells. This conclusion is consistent with our in vivo findings in stem cell transplant patients which provided evidence against significant extra-hepatic production of serum MBL. It provides no ready explanation for the remarkable observation of Mullighan, Heatley, Doherty, Szabo, Grigg, Hughes, Schwarer, Szer, Tait, Bik To and Bardy (2002) Blood 99, 3524-3529 that the presence of variant alleles of mbl-2 in stem cell donors can influence susceptibility to serious infections in their recipients.
SINCE the Royal College's announcement that it would no longer be opposed to non-veterinary surgeons becoming partners in veterinary practices, the scope for the spouses of vets to become involved in ...practices has increased. In this article, Nick Openshaw and Ian Downing examine a number of issues which should be considered before a spouse becomes formally involved in the business - whether as a partner or an employee.
Pooled human anti-Rhesus D antiserum is currently administered for the prevention of RhD alloimmunization. Increased demand, and decreased supply, of donated pooled antiserum has led to the ...investigation of the suitability of human monoclonal anti-RhD antibodies for use in its place. However, it is unclear which biological properties of monoclonal antibodies are important for function in RhD-positive foetal red cell clearance and the prevention of alloimmunization. Various antibodies behave differently in a number of in vitro assays of biological function.
To compare the function and structure of two human anti-RhD IgG1 monoclonal antibodies which differ in their ability to promote red cell lysis in vitro. In particular to examine whether the functional differences correlate to differences in the IgG1 heavy chain constant region (allotype).
We report here the cloning, characterization and re-expression in stable myeloma cell transformants of cDNAs coding for two such antibodies, secreted by the heterohybridoma cell lines ESD-1 (THERAD 03) and LHM 70/45.3 (THERAD 06). The cDNAs were then recombined to exchange portions of the Fc encoding regions and the recombinant antibodies were assayed in vitro to determine RhD-positive red cell-dependent activity.
Recombinant THERAD 03 and 06 antibodies behaved identically to the parent antibodies. The 'inactive' THERAD 06 did not have biological activity reconstituted by exchange with the THERAD 03 Fc regions, nor was THERAD 03 activity abolished by the reciprocal Fc region exchange.
Human monoclonal anti-RhD antibodies can be cloned and re-expressed in stable cell lines, and exhibit identical properties to the parent antibodies. Differences in biological activity cannot be attributed to differences in IgG1 heavy chain allotype.
ABC (ATP-binding-cassette) transporters carry out many vital functions and are involved in numerous diseases, but study of the structure and function of these proteins is often hampered by their ...large size and membrane location. Membrane protein purification usually utilizes detergents to solubilize the protein from the membrane, effectively removing it from its native lipid environment. Subsequently, lipids have to be added back and detergent removed to reconstitute the protein into a lipid bilayer. In the present study, we present the application of a new methodology for the extraction and purification of ABC transporters without the use of detergent, instead, using a copolymer, SMA (polystyrene-co-maleic acid). SMA inserts into a bilayer and assembles into discrete particles, essentially solubilizing the membrane into small discs of bilayer encircled by a polymer, termed SMALPs (SMA lipid particles). We show that this polymer can extract several eukaryotic ABC transporters, P-glycoprotein (ABCB1), MRP1 (multidrug-resistance protein 1; ABCC1), MRP4 (ABCC4), ABCG2 and CFTR (cystic fibrosis transmembrane conductance regulator; ABCC7), from a range of different expression systems. The SMALP-encapsulated ABC transporters can be purified by affinity chromatography, and are able to bind ligands comparably with those in native membranes or detergent micelles. A greater degree of purity and enhanced stability is seen compared with detergent solubilization. The present study demonstrates that eukaryotic ABC transporters can be extracted and purified without ever being removed from their lipid bilayer environment, opening up a wide range of possibilities for the future study of their structure and function.
PDF
