Towards validated multiscale simulations for fusion.

Harnessing energy produced by thermonuclear fusion reactions has the potential to provide a clean and inexpensive source of energy to Earth. However, throughout the past seven decades, physicists learned that creating our very own fusion energy source is very difficult to achieve. We constructed a component-based, multiscale fusion workflow to model fusion plasma inside the core of a tokamak device. To ensure the simulation results agree with experimental values, the model needs to undergo the process of verification, validation and uncertainty quantification (VVUQ). This paper will go over the VVUQ work carried out in the multiscale fusion workflow (MFW), with the help of the EasyVVUQ software library developed by the VECMA project. In particular, similarity of distributions from simulation and experiment is explored as a validation metric. Such initial validation measures provide insights into the accuracy of the simulation results. This article is part of the theme issue 'Reliability and reproducibility in computational science: implementing verification, validation and uncertainty quantification in silico'.

VECMAtk: a scalable verification, validation and uncertainty quantification toolkit for scientific simulations.

We present the VECMA toolkit (VECMAtk), a flexible software environment for single and multiscale simulations that introduces directly applicable and reusable procedures for verification, validation (V&V), sensitivity analysis (SA) and uncertainty quantication (UQ). It enables users to verify key aspects of their applications, systematically compare and validate the simulation outputs against observational or benchmark data, and run simulations conveniently on any platform from the desktop to current multi-petascale computers. In this sequel to our paper on VECMAtk which we presented last year [1] we focus on a range of functional and performance improvements that we have introduced, cover newly introduced components, and applications examples from seven different domains such as conflict modelling and environmental sciences. We also present several implemented patterns for UQ/SA and V&V, and guide the reader through one example concerning COVID-19 modelling in detail. This article is part of the theme issue 'Reliability and reproducibility in computational science: implementing verification, validation and uncertainty quantification in silico'.

Application of the extreme scaling computing pattern on multiscale fusion plasma modelling.

The extreme scaling pattern of the ComPat project is applied to a multi-scale workflow relevant to the magnetically confined fusion problem. This workflow combines transport, turbulence and equilibrium codes (together with additional auxiliaries such as initial conditions and numerical module), which aims at calculating the behaviour of a fusion plasma on long (transport) time scales based on information from much faster (turbulence) time scales. Initial findings of profile measurements are reported in this paper and indicate that, depending on the chosen performance metric for defining 'cost', such as time to completion, efficiency and total energy consumption of the mutliscale workflow, different choices on the number of cores would be made when determining the optimal execution configuration. A variant of the workflow which increases the inherent parallelism is presented, and shown to produce equivalent results at (typically) lower cost compared with the original workflow. This article is part of the theme issue 'Multiscale modelling, simulation and computing: from the desktop to the exascale'.

A GFP promoter fusion library for the study of Salmonella biofilm formation and the mode of action of biofilm inhibitors.

Salmonella, an important foodborne pathogen, forms biofilms in many different environments. The composition of these biofilms differs depending on the growth conditions, and their development is highly coordinated in time. To develop efficient treatments, it is therefore essential that biofilm formation and its inhibition be understood in different environments and in a time-dependent manner. Many currently used techniques, such as transcriptomics or proteomics, are still expensive and thus limited in their application. Therefore, a GFP-promoter fusion library with 79 important Salmonella biofilm genes was developed (covering among other things matrix production, fimbriae and flagella synthesis, and c-di-GMP regulation). This library is a fast, inexpensive, and easy-to-use tool, and can therefore be conducted in different experimental setups in a time-dependent manner. In this paper, four possible applications are highlighted to illustrate and validate the use of this reporter fusion library.

The influence of rejection episodes in recipients of bilateral corneal grafts.

We investigated whether a rejection episode in one graft was associated with rejection in the other graft, in recipients with bilateral corneal transplants. In a prospectively maintained, national register of 14,865 followed corneal grafts, 1476 patients with bilateral penetrating corneal grafts were identified. Occurrence of rejection was a risk factor for graft failure (p < 0.0001). Logistic regression was used to calculate the adjusted odds ratio for rejection in one eye following rejection in the other eye. In the subset of 1118 patients with bilateral grafts but no history of previous grafts or rejections in either eye, the adjusted odds ratio for a rejection episode in the first eye following rejection in the second was 3.27 (95% confidence interval, CI 1.85, 5.79; p < 0.001). The adjusted odds ratio was 2.04 (95% CI 1.07, 3.91; p = 0.03) for rejection in the second eye following rejection in the first. The median time between the first rejection episode in one eye and the first rejection episode in the other eye was 15 months. Patients with bilateral corneal grafts who suffer a graft rejection episode in one eye are at significantly greater odds of suffering a rejection episode in the other corneal transplant.

In vitro adenovirus mediated gene transfer to the human cornea.

BACKGROUND/AIMS: Replication deficient adenovirus is an efficient vector for gene transfer to the cornea. The aim was to optimise the transduction of human corneal endothelium with adenoviral vectors and to measure transgene production from transduced corneas. METHODS: Adenoviral vectors (AdV) encoding enhanced green fluorescent protein (eGFP) or a transgenic protein (scFv) were used to transfect 34 human corneas. Reporter gene expression was assessed after 72-96 hours of organ culture. The kinetics of scFv production was monitored in vitro for 1 month by flow cytometric analysis of corneal supernatants. RESULTS: Transduction of human corneas with high doses (5 x 10(7)-3 x 10(8) pfu) of AdV caused eGFP expression in 12-100% of corneal endothelial cells. Corneas were efficiently transduced following up to 28 days in cold storage. Very high AdV doses (2 x 10(9) pfu) reduced endothelial cell densities to 98 (SD 129) nuclei/mm(2) (compared to 2114 (716) nuclei/mm(2) for all other groups). Transgenic protein production peaked at 2.4 (0.9) microg/cornea/day at 2 weeks post-transduction, and decreased to 1.2 (0.4) microg/cornea/day by 33 days, at which time endothelial cell density had decreased to 431 (685) nuclei/mm(2). CONCLUSION: Human corneas can be efficiently transduced by AdV following extended periods of cold storage, and transgene expression is maintained for at least 1 month in vitro.

Corneal graft rejection occurs despite Fas ligand expression and apoptosis of infiltrating cells.

BACKGROUND/AIMS: Constitutive expression of Fas ligand (CD95L) protects the eye against cell mediated immune responses by inducing apoptosis in infiltrating Fas bearing T cells. This study was designed to examine Fas ligand expression on acutely rejecting rat corneal grafts and to investigate the kinetics of induction of apoptosis in infiltrating leucocytes. METHODS: Orthotopic penetrating corneal transplantation was performed between genetically disparate inbred rats. Fas ligand expression and the phenotype of infiltrating leucocytes were examined by immunohistochemistry. Apoptotic nuclei were visualised in sections of normal rat cornea, rejecting allografts, and time matched isografts by terminal deoxynucleotidyl transferase mediated dUTP biotin nick end labelling (TUNEL) and quantified by video image analysis. Staining with Hoechst dye 33258 was used to confirm the presence of apoptotic nuclei. RESULTS: Fas ligand was expressed on corneal endothelial and epithelial cells during acute corneal graft rejection. At all time points examined, including as early as the fifth postoperative day, the cells infiltrating both corneal isografts and allografts were TUNEL positive. By the 15th postoperative day, over 90% of all nuclei, many of which were T cells, were apoptotic. CONCLUSION: Expression of Fas ligand is not downregulated on the cornea during allograft rejection and infiltrating leucocytes in both isografts and allografts die rapidly in situ. Despite the death of the cells believed to be responsible for rejection, isografts survive indefinitely whereas allografts are irreparably damaged.

Influence of format on in vitro penetration of antibody fragments through porcine cornea.

AIM: Antibody fragments, appropriately formulated, can penetrate through the ocular surface and thus have potential as therapeutic agents. The aim was to investigate the influence of protein fragment format on the kinetics and extent of ocular penetration in vitro. METHODS: Immunoglobulin single chain variable domain fragments of a murine monoclonal antibody with specificity for rat CD4 were engineered with a 20 or 11 amino acid linker by assembly polymerase chain reaction, expressed in Escherichia coli and purified by chromatography. Fab fragments of the parental antibody were prepared by papain digestion. Antibody fragments were formulated with a penetration and a viscosity enhancer and were applied to the surface of perfused pig corneas for up to 10 hours in vitro. Penetration was quantified by flow cytometry on rat thymocytes. RESULTS: 20-mer antibody fragments formed natural monomers and dimers following purification that could be separately isolated, while 11-mer fragments were dimeric. All formats of fragment (20-mer monomers and dimers, 11-mer dimers, Fab) showed penetration through the pig cornea after 6 hours of intermittent topical administration. CONCLUSION: Antibody fragments of different shapes and sizes can penetrate the cornea after topical administration, thereby increasing the potential of this class of proteins for topical ophthalmic use.

The role of resident accessory cells in corneal allograft rejection in the rabbit.

Corneal grafts are more likely to be rejected when placed in a vascularized rather than in a normal host cornea. Using immunohistochemical techniques, normal rabbit cornea was found to contain measurable numbers of cells of hemopoietic origin, probably of either macrophage or dendritic lineage. After the deliberate induction of corneal inflammation and neovascularization, the number of these accessory cells was found to increase significantly. There was also a marked increase in the number of T cells present. Enzyme staining indicated a degree of heterogeneity in the infiltrate. The process of rejection of rabbit corneal grafts was found to occur earlier when additional infiltrating cells were present in either donor button or graft bed, and earlier still when the load of infiltrating cells was increased in both donor and recipient. We hypothesize that resident accessory cells of recipient origin may be implicated in graft rejection in vascularized, inflamed corneas.

Histocompatibility antigen and passenger cell content of normal and diseased human cornea.

The outcome of clinical corneal transplantation depends on the degree of vascularization and inflammation present in the graft bed at the time of the operation, but the reason for this is unclear. Normal, diseased, and rejected human corneas have been examined with an immunoperoxidase staining procedure, employing monoclonal antibodies to class I and II major histocompatibility complex (MHC) antigens and to other leukocyte markers. In particular, departures from normal in the expression of MHC antigens and in the passenger cell distribution in the diseased or rejected corneas were sought. MHC antigen expression did not alter with inflammation, vascularization, or rejection. However, dendritic-like passenger cells, which were found in low numbers throughout the central stroma of normal cornea as well as in basal epithelium, significantly increased in number in vascularized corneas. We suggest that the breakdown of corneal privilege in vascularized eyes may reflect the increased number of accessory cells in the graft bed.

A comparison of the effects of topical cyclosporine and topical steroid on rabbit corneal allograft rejection.

A comparison was made of the effects of topically applied cyclosporine, and topically applied prednisolone acetate, on the prolongation of corneal allograft survival in a recently developed prevascularized rabbit eye model. Animals were treated four times daily for 28 days postgrafting. Both drugs prolonged graft survival when compared with placebo or no treatment but the corticosteroid was significantly more effective than cyclosporine. Furthermore, anterior segment inflammation and graft vascularization were considerably less marked in animals treated with steroid. No cyclosporine could be detected by radioimmunoassay in anterior chamber fluid removed by paracentesis from grafted animals treated with cyclosporine, suggesting poor absorption of the drug across the cornea.

Prolongation of sheep corneal allograft survival by ex vivo transfer of the gene encoding interleukin-10.

BACKGROUND: Modification of a donor cornea by gene therapy ex vivo has potential to modulate irreversible rejection, the major cause of corneal graft failure. Our aim was to transfer the gene encoding mammalian IL-10 to ovine donor corneas and to determine subsequent orthotopic corneal allograft survival in an outbred sheep model. METHODS: The replicative capacity of ovine corneal endothelium was determined by autoradiography after deliberate injury. A replication-defective adenovirus was used to deliver the lacZ reporter gene to ovine corneas and transfected corneas were organ-cultured in vitro to allow transfection efficiency, duration of reporter gene expression, and toxicity attributable to the vector to be determined. A cDNA encoding full-length ovine IL-10 was cloned into an adenoviral vector that was used to transfect donor corneas ex vivo before transplantation. Orthotopic penetrating corneal transplantation was performed in outbred sheep. RESULTS: Sheep corneal endothelium was found to be essentially amitotic. Transfection of > 70% corneal endothelial cells was achieved with the viral vector and expression was maintained for 28 days in vitro. IL-10 mRNA was detectable in transfected, organ-cultured corneas for 21 days in vitro. Donor corneas transfected with cDNA encoding IL-10 showed significantly prolonged survival after penetrating keratoplasty (median 55 days, range 19 > or =300 days) compared with control corneas (median 20.5 days, range 18-32 days, P=0.011). CONCLUSION: Local gene therapy-mediated expression of the immunomodulatory cytokine IL-10 has the potential to reduce the incidence of corneal graft rejection and to prolong corneal allograft survival.

Survival of rabbit limbal stem cell allografts.

Failure of a specialized population of corneal epithelial stem cells found in the peripheral cornea and limbus results in ocular surface disease, which may be amenable to treatment by transplantation of limbal tissue. This study was designed to investigate donor limbal stem cell allograft survival in rabbits with ocular surface disease. Rabbits underwent corneal epithelial debridement and limbal ablation to induce ocular surface disease and were then treated by limbal stem cell allotransplantation, by allotransplantation plus topical steroid, or by topical steroid only (n = 7 for each group). Donors and recipients were sex mismatched. Recipients were followed for up to 5 months. Outcome was assessed by daily slit-lamp examination, weekly impression cytology and photographic record, end-point sex chromatin and fluorescent cell tracer analyses, histology, and immunohistochemistry. In no case was a completely normal ocular surface regained, but some animals that received grafts plus corticosteroids fared best by all criteria used. In the absence of immunosuppression, graft hemorrhagia (believed to be a manifestation of graft rejection) occurred within the first month, the cornea became resurfaced with conjunctiva-derived cells, and no donor cells survived centrally in the long term. Topical corticosteroids reduced the number and severity of these episodes significantly, and were associated with survival of some donor-derived cells in the central cornea of some grafted animals. Thus, rabbit limbal stem cell allografts appeared to undergo rejection, which could be modified by immunosuppression, but useful regeneration of the ocular surface occurred only where rejection was circumvented.

Patterns of corneal graft rejection in the rabbit and reversal of rejection with monoclonal antibodies.

The purpose of this study was to determine whether the local administration of monoclonal antibodies could reverse rabbit corneal graft rejection. To provide a rational basis for the choice of monoclonal antibodies as potential immunosuppressive agents, the phenotypes of cells infiltrating rejecting rabbit corneal allografts were examined by immunohistochemistry. About half the leukocytes accumulating in these grafts bore an immunodominant T cell marker, over two-thirds carried MHC class II antigens, and about one-fifth carried myeloid cell markers. A kinetic study of the cell population appearing in rabbit aqueous during corneal graft rejection was performed by examination of repetitive anterior chamber taps taken over a ten-day period; again, the major components were T cells, MHC class II antigen-positive cells and myeloid cells. Monoclonal antibodies L11/135 (directed against a peripheral T cell determinant), 2C4 (directed against a monomorphic MHC class II antigen), and LION 2 (directed against a myeloid antigen) were chosen for intracameral injection into rabbits with rejecting corneal grafts. Each animal received a total of 50-100 micrograms of antibody in two injections at 3-4-day intervals. L11/135 and LION 2 reversed rejection in 5/9 and 8/12 animals, respectively, in the absence of any other immunosuppression; 2C4 was without effect. We suggest that monoclonal antibody therapy in corneal transplantation deserves further attention.

Penetrating corneal transplantation in the inbred rat: a new model.

A model of orthotopic penetrating keratoplasty has been developed in the inbred rat using both avascular and prevascularized recipient beds. The surgical procedure is conventional and can be achieved with standard instrumentation. Isografts into avascular recipient beds (Fisher 344 into Fisher 344 strain combination) were successful and survived indefinitely with excellent corneal function judged either visually by clarity and lack of oedema, or histologically at autopsy. Allografts into avascular beds (DA into Fisher 344 strain combination) became cloudy and oedematous at a median of day 12 postgraft; 43% spontaneously recovered clarity while the remaining 57% remained opaque or became scarred. Penetrating grafts also were performed in eyes prevascularized by the placement of sutures approximately 3 weeks prior to transplantation. Most isografts into prevascularized and inflamed beds underwent a transient episode of oedema, which quickly resolved and was felt to result from postsurgical inflammation. All allografts into prevascularized beds became oedematous and cloudy; 76% went on to fail completely, while 24% cleared without treatment. End-point histology showed normal graft morphology in the isografts; failed allografts showed a picture consistent with immunologic rejection. The model, which allows corneal transplantation to be performed against a constant histocompatibility barrier, may be useful in studies of rejection.

Endothelial repair in the rat cornea.

Destruction of the central endothelium of the rat cornea was produced by mechanical injury, total debridement, or transcorneal freezing. Endothelial repair was then studied using specular microscopy, histological staining, pachymetry, and autoradiographic analysis of the incorporation of tritiated thymidine into nuclear DNA. Following an initial process of cell slide to cover the endothelial defect, extensive cellular division occurred at the margins of the wound, with approximately 45% of cells in the wound area showing incorporation of tritiated thymidine. An intact monolayer of irregularly shaped cells was reestablished by 2-14 days, depending on the wound. These results suggest that the corneal endothelial repair processes in the rat are more analogous to those of the rabbit than to those of the cat or primate.

Effect of inflammation on antibiotic penetration into the anterior segment of the rat eye.

Bacterial infections were established in the right cornea of rats. Animals infected with Staphylococcus aureus were given cephradine intravenously (IV) (40 mg/kg) or topically (50 mg/ml) to both eyes. Animals infected with Pseudomonas aeruginosa were given gentamicin sulfate IV (40 mg/kg) or topically (10 mg/ml). Antibiotic concentrations in cornea and aqueous humor were measured for 4 hrs following dosing using bioassay and radioimmunoassay. In general, infection significantly increased the concentrations obtained soon after dosing. Topically applied cephradine passed through infected eyes more quickly than through normal eyes. Of the pharmacokinetic parameters derived, the permeability of the corneal epithelium to gentamicin in the rat more closely agrees with reported human values than does the rabbit, while the coefficient of elimination from aqueous in the rat is considerably greater than that for either humans or rabbits. This suggests that there are both advantages and disadvantages in using the rat for therapeutic studies of ocular disease.

Enzymatic disaggregation of the infected rat cornea.

Analysis of cell populations in the cornea may be performed rapidly and accurately employing the technique of enzymatic disaggregation. To illustrate this method normal rat corneas and corneas infected 24 and 48 hours previously with Staphylococcus aureus were disaggregated in a solution containing pancreatin and collagenase. The cells released were counted and identified morphologically. These results were compared to cell counts made from histologic sections. Over 95% of the corneal cells were viable after the disaggregation and leukocytes obtained from the infected corneas retained their phagocytic capacity. This approach allows sensitive analysis of cell populations in a wide range of corneal conditions, including infection and allograft rejection.

Mice deficient in tumor necrosis factor receptors p55 and p75, interleukin-4, or inducible nitric oxide synthase are susceptible to endotoxin-induced uveitis.

PURPOSE: To investigate the roles of tumor necrosis factor-alpha (TNF-alpha), interleukin-4 (IL-4), and inducible nitric oxide synthase (iNOS) in endotoxin-induced uveitis (EIU) using gene knock-out mice. METHODS: Mice (C57BL/6 x 129) either of normal phenotype or deficient in the genes encoding one or both tumor necrosis factor receptors (TNFR p55 and TNFR p75), IL-4, or iNOS were given footpad injections of 400 micrograms Escherichia coli lipopolysaccharide. Animals were killed 24 hours later, and infiltrating cells were counted on 5-micron ocular cross-sections through the optic nerve. RESULTS: All abnormal mouse phenotypes were susceptible to EIU. Yet, TNFR p55 and IL-4 gene knock-out mice experienced less ocular inflammation than control animals (P = 0.021 and 0.007, respectively), whereas disease was not reduced for iNOS-deficient mice. Mice deficient in TNFR p55 and TNFR p75 experienced milder EIU than mice lacking TNFR p75 alone (P = 0.046). CONCLUSIONS: Mice deficient in TNFR p55 and TNFR p75, IL-4, or iNOS retain the susceptibility to EIU, but TNF-alpha and IL-4 influence the influx of inflammatory cells to the eye during this disease.