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Cutting strategy research has traditionally been focused on hard materials that are intrinsically difficult to machine. An increase in the desire for personalized products has led to the requirement of the direct machining of polymers for personalized products. Little research is evident in the literature on the analysis of optimal machining parameters for machining materials such as polypropylene. One of the vital factors that affects the quality of polypropylene products and the respective machining strategy is surface roughness. This research is aimed at extracting information on the machining of polypropylene materials. A surface roughness predictive model based on neural networks has been developed. The design of experiments approach is used to obtain an adequate predictive model for the process planning which is further utilized as an input to the predictive model. The model mainly hinges on three independent variables namely spindle speed, feed rate, and depth of cut. Extensive experimental work on different network topologies and training algorithms has been performed to predict the behaviour of the surface roughness for machined polypropylene products. The results illustrate the benefits of being able to determine surface roughness values. This allows for the determination of optimal cutting strategies and tooling for the required surface roughness. The performance predictive model has been found to be satisfactory over the dataset for polypropylene machining. Hypothesis testing has also been carried out to identify the confidence of the predictive model.

With today's highly competitive global manufacturing marketplace, the pressure for right-first-time manufacture has never been so high. New emerging data standards combined with machine data collection methods, such as in-process verification lead the way to a complete paradigm shift from the traditional manufacturing and inspection to intelligent networked process control. Low-level G and M codes offer very limited information on machine capabilities or work piece characteristics which consequently, results in no information being available on manufacturing processes, inspection plans and work piece attributes in terms of tolerances, etc. and design features to computer numerically controlled (CNC) machines. One solution to the aforementioned problems is using STEP-NC (ISO 14649) suite of standards, which aim to provide higher-level information for process control. In this paper, the authors provide a definition for process control in CNC manufacturing and identify the challenges in achieving process control in current CNC manufacturing scenario. The paper then introduces a STEP-compliant framework that makes use of self-learning algorithms that enable the manufacturing system to learn from previous data and results in eliminating the errors and consistently producing quality products. The framework relies on knowledge discovery methods such as data mining encapsulated in a process analyser to derive rules for corrective measures to control the manufacturing process. The design for the knowledge-based process analyser and the various process control mechanisms conclude the paper.

Manufacturing firms are seeking more efficient methods of CNC manufacture. ISO14649 informally known as STEP-NC has been proposed as a high-level hierarchical manufacturing information model as a replacement for the low-level machining instructions of ISO6983 and RS274D. In this paper, the applicability of STEP-NC as an enabler for creating an adaptive global manufacturing system is examined. The overall framework of the system is presented followed by an outline of its information requirements. Suitability of STEP-NC to support each requirement is then studied with the necessary additions highlighted. Finally, a test component is used in conjunction with a prototype of the advanced global manufacturing system to demonstrate the applicability of the STEP-NC standard to support manufacturing information in such a system.

As the ratio of transport costs to total product cost increases, manufacturers try to fulfil the demands of each geographical region with products manufactured within the same region. Since the manufacturing resources available at each venue can be varied, it is necessary to adapt the manufacturing process plan for each production facility. Traditionally, this process is carried out manually by engineers utilizing computer-aided design (CAD) files. With the advances in artificial intelligence technology and emergence of integration standards such as STEP, it is now possible to automate the CAD/computer-aided manufacturing (CAM)/computer numerical control (CNC) interface. Distributed artificial intelligence techniques and mobile agents in particular can be used to transfer information throughout the manufacturing network and construct the distributed knowledge-base required for the intelligent interoperable integration of product data models and manufacturing resources. This paper explores the application ...

For many years, manufacturing firms have been seeking more efficient ways of manufacturing components with CNC machines. The emerging standards ISO 14649 and ISO 10303 (AP238) present an opportunity to revolutionize the way CNC machines are traditionally programmed. These standards better known as STEP-NC replace the traditional tool movement description languages with hierarchical data structures that allow a new breed of CNC to store part geometry together with the working steps of the operations required to manufacture the part. STEP-NC provides the ability to store and utilise high level and detailed information from the CAD system to the intelligent STEP compliant CNC controller. With the advent of STEP-NC, computer aided process planning has become a critical link in the CAx process chain with the major requirement to generate interoperable process plans. The authors therefore believe it is necessary to redefine CAPP to reflect the change from the traditional tool movement based programming to STEP-NC based programming. This paper examines the application of distributed artificial intelligence methods, namely collaborative multi-agent systems in designing an object-oriented process planning system for prismatic components in a STEP-NC compliant environment. The specification and design of a prototype system entitled the Multi-Agent System for Computer Aided Process Planning (MASCAPP) is outlined. Two test components have been designed, process planned, simulated on the machine controller and finally machined, to demonstrate the capabilities of the system and illustrate the activities required to implement STEP compliant manufacturing.

CNC manufacturing has evolved through the use of faster, more precise and more capable CNC controllers and machine tools. These enhancements in machine tools however have not been integrated under a common platform to support CAD/CAM/CNC software inter-operability and as a result a plethora of standards is being used for these systems. ISO10303 (STEP) and ISO14649 (STEP-NC) seek to eliminate the barriers in the exchange of information in the CNC manufacturing chain and enable inter-operability throughout the manufacturing software domain. This paper introduces a novel software platform called the Integrated Platform for Process Planning and Control (IP3AC) to support the rapid development of STEP-NC compliant CNC manufacturing software.

Over the last five years, a great deal of research effort has been concentrated on the development of a new data model ISO 14649, informally known as STEP-NC. It has been strongly argued that STEP-NC has huge implications on the integration of the computer-aided design/computer-aided process planning/computer-aided manufacture (CAD/CAPP/CAM) (CAx) systems, giving the opportunity to realize interoperable computer numerical control (CNC) manufacturing. This is largely owing to the data model, which provides the capability to revolutionize the current state of the art in CNC manufacturing by offering a bi-directional interface with a high-level description of the geometrical and manufacturing information. This paper provides a view of how these STEP-NC compliant information models can be used to support the wire electrical discharge machining (WEDM) CAD to the CNC process chain. The models are based on part 13 of the ISO 14649 standard, which is dedicated to the WEDM process, together with part 10 of the standard, which specifies the general machining information. The information models have been identified and their structures have been defined and modelled using the unified modelling language (UML). A STEP-NC compliant WEDM CAx prototype system, based on the Java and the object-oriented database management system (DBMS) ObjectStore, has been constructed with an example case study to demonstrate the information models.

Ethylene vinyl acetate (EVA) is a highly utilised material for the manufacture of shoe soles and mid-soles due to its comfort, durability and UV stability. It is also used extensively for exercise mats, wall insulation and sports protection padding. The material can be manufactured into sheets by compression moulding or injection-moulding processes. This paper is concerned with research to gain knowledge of the behaviour of EVA during the injection moulding of shoe soles and shoe mid-soles. Though there is significant experience with the general use of EVA during the injection-moulding process this knowledge has been gained through years of experience and moulds are designed by a combination of the producer's knowledge and trial and error. With the increase in demand for customisation of mass-produced products a method for automating the design of moulds for expanding EVA products is highly desirable. When mixed with a suitable blowing agent, EVA has the remarkable ability to expand between 30% and 90% compared to the injection mould size, depending on the amount of blowing agent added. However, EVA does not expand uniformly and currently there is no precise method of predicting the expansion of components with variable thickness. This paper presents a methodology for determining the expansion characteristics of EVA coupled with results relating to the initial findings for material expansion to enable the design of a method for automatically generating mould cavity geometry.

Computer aided part programming is an information intensive activity, and provides an interesting area for the application of artificial intelligence, knowledge-based systems and recently for agent technologies. Intelligent software agents can behave and respond autonomously to set criteria. The flexibility of agents and their ability to make autonomous/cooperative decisions makes them an excellent tool in the development of future computer aided process planning systems. In addition, a new ISO standard for NC machine tool programming is emerging called ISO 14649, informally known as STEP-NC. This standard is aimed to replace the existing ISO 6983 (G&M code) standard, which has been in use since the first NC machine tools were developed in the 1950s. STEP-NC does not describe tool movements but uses a higher-level language to define operations known as Workingsteps. A new breed of intelligent controller is envisaged to determine optimized tool trajectories allowing advanced inter-operability and a bi-dire...

Wire electrical discharge machining (WEDM) is a specialised thermal machining process capable of accurately machining parts with varying hardness or complex shapes, which have sharp edges that are very difficult to be machined by the main stream machining processes. This practical technology of the WEDM process is based on the conventional EDM sparking phenomenon utilising the widely accepted non-contact technique of material removal. Since the introduction of the process, WEDM has evolved from a simple means of making tools and dies to the best alternative of producing micro-scale parts with the highest degree of dimensional accuracy and surface finish quality. Over the years, the WEDM process has remained as a competitive and economical machining option fulfilling the demanding machining requirements imposed by the short product development cycles and the growing cost pressures. However, the risk of wire breakage and bending has undermined the full potential of the process drastically reducing the efficiency and accuracy of the WEDM operation. A significant amount of research has explored the different methodologies of achieving the ultimate WEDM goals of optimising the numerous process parameters analytically with the total elimination of the wire breakages thereby also improving the overall machining reliability. This paper reviews the vast array of research work carried out from the spin-off from the EDM process to the development of the WEDM. It reports on the WEDM research involving the optimisation of the process parameters surveying the influence of the various factors affecting the machining performance and productivity. The paper also highlights the adaptive monitoring and control of the process investigating the feasibility of the different control strategies of obtaining the optimal machining conditions. A wide range of WEDM industrial applications are reported together with the development of the hybrid machining processes. The final part of the paper discusses these developments and outlines the possible trends for future WEDM research.

Data, information and knowledge are very important commodities for organisations. The effective utilisation of these 'commodities' is increasingly the only way for organisations to achieve and sustain competitive advantage. In the field of mechanical engineering there are vast numbers of information and knowledge sources that are utilised throughout the design of an artefact or system. These may include documentation, component catalogues, past designs, new technologies, complex methodologies as well as a whole range of informal and formal sources developed through discussions and meetings. The effective utilisation and application of these information and knowledge commodities help enable the generation of feasible design alternatives and assist the decision-making process, which ultimately determines the success of the designed artefact. This paper discusses data, information and knowledge, providing formal definitions and an understanding of the relations and limitations of these resources. This understanding enables the development of better mechanisms and procedures for the capture and reuse of information and knowledge in engineering design. In particular, the approach of this work is to consider the intended reuse and level of application for knowledge in order to determine the requirements for its acquisition. Using this approach, an overall framework for the requirements of capturing, storing and reusing information and knowledge in engineering design is generated.

Graft-versus-host disease (GVHD) is a major complication of bone marrow transplantation that can occur in either acute or chronic forms. Much of the long-term pathology seen in chronic GVHD is a result of autoantibody production. In the DBA/2-->B6D2F1 murine model of chronic GVHD, anti-ssDNA autoantibodies can be detected by 14 days post cell transfer. These autoantibodies are not observed in B6D2F1 recipients of cells from C57BL/6 or B10.D2 donors, which develop acute rather than chronic GVHD. Therefore, in this model, donor genetic factors predispose to the development of chronic GVHD in recipients. We performed a genetic analysis aimed at mapping donor loci that influence the magnitude of early autoantibody production in B6D2F1 recipients of cells from DBA/2 donor mice. Linkage analysis suggested an influence of two loci: a locus on chromosome 11 linked to D11Mit278 and a locus on chromosome 4 linked to D4Mit226. The locus on chromosome 11 also appeared to influence the development of renal pathology associated with chronic GVHD.

Graft-versus-host disease (GVHD) is the major complication occurring after bone marrow transplantation. The severity of GVHD varies widely, with this variation generally being attributed to variation in the degree of disparity between host and donor for minor histocompatibility antigens. However, it is also possible that other forms of polymorphism, such as polymorphisms in immune effector molecules, might play a significant role in determining GVHD severity. In order to investigate this hypothesis, we are studying the genetic factors that influence GVHD development in a murine model. We here report the first results of this analysis, which demonstrate that a locus on Chromosome 1 of the mouse, and possibly also a locus on Chromosome 4, exert considerable influence over the development of one aspect of acute GVHD - splenomegaly - in a parent--F1 murine model. These results demonstrate that non-MHC genes can exert quite significant effects on the development of GVHD-associated pathology and that gene mapping can be used as a tool to identify these loci. Further analysis of such loci will allow identification of the mechanism whereby they influence GVHD and may lead in the future to improved selection of donors for human bone marrow transplantation.

Phosphatidic acid and its derivatives play potentially important roles as extracellular messengers in biological systems. An ecto-phosphatidic acid phosphohydrolase (ecto-PAPase) has been identified which effectively regulates neutrophil responses to exogenous phosphatidic acid by converting the substrate to diacylglycerol. The present study was undertaken to characterize this ecto-enzyme on intact cells and to isolate the enzyme from solubilized neutrophil extracts. In the absence of detergent, short chain phosphatidic acids were hydrolysed most effectively by neutrophil plasma membrane ecto-PAPase; both saturated and unsaturated long chain phosphatidic acids were relatively resistant to hydrolysis. Both long (C18:1) and short (C8) chain lyso-phosphatidic acids were hydrolysed at rates comparable with those observed for short chain (diC8) phosphatidic acid. Activity of the ecto-enzyme accounted for essentially all of the N-ethylmaleimide-insensitive, Mg2+-independent PAPase activity recovered from disrupted neutrophils. At 37 °C and pH 7.2, the apparent Km for dioctanoyl phosphatidic acid (diC8PA) was 1.4×10-3 M. Other phosphatidic acids and lysophosphatidic acids inhibited hydrolysis of [32P]diC8PA in a rank order that correlated with competitor solubility, lysophosphatidic acids and unsaturated phosphatidic acids being much more effective inhibitors than long chain saturated phosphatidic acids. Dioleoyl (C18:1) phosphatidic acid was an unexpectedly strong inhibitor of activity, in comparison with its ability to act as a direct substrate in the absence of detergent. Other inhibitors of neutrophil ecto-PAPase included sphingosine, dimethyl- and dihydro-sphingosine, propranolol, NaF and MgCl2. Of several leucocyte populations isolated from human blood by FACS, including T cells, B cells, NK lymphocytes and monocytes, ecto-PAPase was most prevalent on neutrophils; erythrocytes were essentially devoid of activity. A non-hydrolysable, phosphonate analogue of phosphatidic acid, phosphonate 1, efficiently solubilized catalytic activity from intact neutrophils without causing cell disruption or increasing permeability. Enzyme activity in solubilized extracts was purified in the absence of detergent by successive heparin–Sepharose, gel filtration and anion exchange chromatography. By assaying activity in renatured SDS/polyacrylamide gel slices, the molecular mass of neutrophil ecto-PAPase was estimated to be between 45 and 52 kDa, similar to the molecular mass of previously purified plasma membrane PAPases. Since a large portion of neutrophil plasma membrane PAPase is available for hydrolysis of exogenous substrates, ecto-PAPase may play an important role in regulating inflammatory cell responses to extracellular phosphatidic acid in biological systems.

Strains of Listeria monocytogenes serotype 4b account for a large fraction of sporadic listeriosis cases, as well as all major food-borne epidemics attributed to this pathogen. We have identified a set of three monoclonal antibodies which showed a high degree of specificity for strains of L. monocytogenes serotype 4b. Two of these antibodies (c74.33 and c74.180, isotypes immunoglobulin M [IgM] and IgG3, respectively) recognized all serotype 4b strains, whereas antibody c74.22 (isotype IgG1) failed to recognize certain epidemic-associated strains. The corresponding antigens were located on the surface of the bacteria and were expressed following bacterial growth in different media and over a wide range of temperatures (4, 22, and 37 degrees C). Heating L. monocytogenes cells at 80,90, or 100 degrees C abolished reactivity for c74.22 but not for c74.33 MAb. These MAbs were negative for all of the non-Listeria strains tested, including representatives of several gram-negative and gram-positive species. The surface antigen recognized by c74.22 appeared to be associated with the ability of the bacteria to enter (invade) mammalian cells in culture.

A new technique using oxygen plasma etching was developed for studying the dispersion of filler particles in polymer solids. With this method, the organic polymer matrix is selectively etched from the top surface down into the bulk, revealing the dispersion state of the filler particles upon scanning electron microscopic analysis. A polymer composite, comprised of zinc oxide particles and a linear styrene-n-butylmethacrylate copolymer, was used to investigate the particle dispersion as a function of filler volume fraction. Preliminary data on the effect of a surfactant on filler dispersion is also discussed

Among the many factors that limit crop productivity world-wide , unfavorable environmental challenges caused by nonliving (abiotic) factors are likely to be the most critical. Human population growth and the resulting increase in demand for agricultural products, progressive loss of arable land and water resources, and climatic instability will make it increasingly difficult to meet these needs. One part of the solution to this problem will be to create new crop varieties that are able to maintain high productivity under suboptimal environmental conditions. Clearly, improvements will continue to be made through crop breeding, but it is likely that new strategies will have to be developed to double global agricultural production from a fixed amount of land with no increase in water or other inputs. These strategies will depend on a thorough knowledge of the molecular mechanisms used by plants to acclimate to harsh environmental conditions. In this chapter, I review the rapid progress in recent years in our understanding of these processes, which has come through research in model plant species such as Arabidopsis, and outline the ongoing translation of these results to crop plants, including cotton. Expression of protective genes and modification of the regulatory mechanisms that control the expression of these genes in response to abiotic stress hold significant promise for future agricultural systems around the world.

Bacteroides (Porphyromonas) gingivalis, which has been implicated as an etiologic agent in human periodontal diseases, has been shown to bind and degrade human fibrinogen. B. gingivalis strains bind fibrinogen reversibly and with high affinity and bind to a specific region of the fibrinogen molecule that appears to be located between the D and E domains (M. S. Lantz, R. D. Allen, P. Bounelis, L. M. Switalski, and M. Hook, J. Bacteriol. 172:716-726, 1990). We now report that human fibrinogen is bound and then degraded by specific B. gingivalis components that appear to be localized at the cell surface. Fibrinogen binding to bacterial cells occurred at 4, 22, and 37 degrees C. A functional fibrinogen-binding component (Mr, 150,000) was identified when sodium dodecyl sulfate-solubilized bacteria were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and probed with 125I-fibrinogen. Fibrinogen degradation did not occur at 4 degrees C but did occur at 22 and 37 degrees C. When bacteria and iodinated fibrinogen were incubated at 37 degrees C, two major fibrinogen fragments (Mr, 97,000 and 50,000) accumulated in incubation mixture supernatant fractions. Two major fibrinogen-degrading components (Mr, 120,000 and 150,000) have been identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in substrate-containing gels. Fibrinogen degradation by the Mr-120,000 and -150,000 proteases was enhanced by reducing agents, completely inhibited by N-alpha-p-tosyl-L-lysyl chloromethyl ketone, and partially inhibited by n-ethyl maleimide, suggesting that these enzymes are thiol-dependent proteases with trypsinlike substrate specificity. The fibrinogen-binding component could be separated from the fibrinogen-degrading components by selective solubilization of bacteria in sodium deoxycholate.

Homozygosity for either of the lymphoproliferation (lpr) or generalized lymphoproliferative disease (gld) mutations of mice causes the development of systemic lupus erythematosus-like autoimmune syndromes that are characterized by severe lymphadenopathy and highly elevated serum immunoglobulin levels. Although the mutations are nonallelic, analysis of homozygous lpr/lpr and gld/gld mice on the same strain background has indicated that the pathology and severity of the autoimmune syndromes induced by these mutations are indistinguishable. To explain this, it has previously been suggested that lpr and gld may represent mutations in molecules involved in sequential steps of an intracellular metabolic pathway of T cells. We have now investigated the behavior of both lpr and gld in a variety of bone marrow chimeras and have found that functional differences between lpr and gld become apparent after bone marrow transfer. Transfer of lpr/lpr bone marrow to irradiated congenic +/+ recipients caused the development of a graft-vs.-host-like lymphoid wasting syndrome, whereas transfer of gld/gld bone marrow to +/+ recipients resulted in development of a gld-like autoimmune syndrome. Additionally, gld/gld hosts behaved like +/+ hosts irrespective of the genotype of the donor bone marrow, whereas lpr/lpr hosts behaved unlike +/+ hosts when reconstituted with either lpr/lpr, gld/gld, or +/+ bone marrow. These are the first clear differences between these two mutations yet described. Our studies indicate that the molecule altered by the gld mutation is expressed only by bone marrow-derived cells, whereas the molecule altered by the lpr mutation is expressed by both bone marrow-derived cells and by one or more peripheral radioresistant cell populations. To reconcile these differences with the fact that homozygous lpr/lpr and gld/gld mice are indistinguishable, we suggest an alternative model for the relationship between the lpr and gld mutations in which the two molecules affected represent an interacting ligand-receptor pair expressed by different cells.

A cDNA clone of a wheat germin-like oxalate oxidase (OxO) gene regulated by the constitutive CaMV 35S promoter was expressed in a hybrid poplar clone, Populus x euramericana ('Ogy'). Previous studies showed that OxO is likely to play an important role in several aspects of plant development, stress response, and defense against pathogens. In order to study this wheat oxalate oxidase gene in woody plants, the expression of this gene and the functions of the encoded enzyme were examined in vitro and in vivo in transgenic 'Ogy'. The enzyme activity in the transformed 'Ogy' was visualized by histochemical assays and in SDS-polyacrylamide gels. It was found that the wheat OxO gene is expressed in leaves, stems, and roots of the transgenic 'Ogy' plants and the encoded enzyme is able to break down oxalic acid. Transgenic 'Ogy' leaves were more tolerant to oxalic acid as well as more effective in increasing the pH in an oxalic acid solution when compared to untransformed controls. In addition, when leaf disks from 'Ogy' plants were inoculated with conidia of the poplar pathogenic fungus Septoria musiva, which produces oxalic acid, the OxO-transformed plants were more resistant than the untransformed controls.

The contractile vacuole (CV) is an osmoregulatory organelle whose mechanisms of function are poorly understood. Immunological studies in the last decade have demonstrated abundant proton-translocating V-type ATPases (V-ATPases) in its membrane that could provide the energy, from proton electrochemical gradients, for moving ions into the CV to be followed by water. This review emphasizes recent work on the contractile vacuole complex (CVC) of Paramecium including (1) CV expulsion, (2) a role for V-ATPases in sequestering fluid, (3) identifying ions in the cytosol and in the CV, (4) in situ electrophysiological parameters of the CVC membrane, and (5) a better understanding of the membrane dynamics of this organelle.

Cellular membranes are made in a cell's biosynthetic pathway and are composed of similar biochemical constituents. Nevertheless, they become differentiated as membrane components are sorted into different membrane-limited compartments. We summarize the morphological and immunological similarities and differences seen in the membranes of the various interacting compartments in the single-celled organism, Paramecium. Besides the biosynthetic pathway, membranes of the regulated secretory pathway, endocytic pathway, and phagocytic pathway are highlighted. Paramecium is a multipolarized cell in the sense that several different pools of membrane-limited compartments are targeted for exocytosis at very specific sites at the cell surface. Thus, the method used by this cell to sort and package its membrane subunits into different compartments, the processes used to transport these compartments to specific locations at the plasma membrane and to other intracellular fusion sites, the processes of membrane retrieval, and the processes of membrane docking and fusion are reviewed. Paramecium has provided an excellent model for studying the complexities of membrane trafficking in one cell using both morphological and immunocytochemical techniques. This cell also promises to be a useful model for studying aspects of the molecular biology of membrane sorting, retrieval, transport, and fusion.

The relationship between acute and chronic graft-versus-host disease (GVHD) is not well understood. A murine model of acute and chronic GVHD is the B6D2F1 parent → F1 model in which transfer of C57BL/6 parental strain lymphoid cells to B6D2F1 recipients results in development of Th1-mediated acute GVHD, whereas transfer of DBA/2 parental strain lymphoid cells to B6D2F1 recipients results in development of Th2-mediated chronic GVHD. Numerous studies have investigated the reason for the differential development of acute versus chronic GVHD in this model but have as yet failed to identify the factor that determines which type of T helper cell will predominate and thereby which type of GVHD will develop. In this report, we demonstrate, using congenic strains of mice, that a locus in the vicinity of the Mtv7 locus on Chromosome 1 of the mouse significantly influences development of acute versus chronic GVHD in the B6D2F1 model.

Cardiovascular and cerebrovascular disease are major causes of morbidity and mortality after kidney transplantation. The aim of this longitudinal study was to examine the natural history of carotid plaque and to determine risk factors for the progression of vascular disease in uremic, type 1 diabetic patients who received a combined kidney and pancreas transplant.Carotid artery (n=765) and lower limb vascular duplex scanning (n=656) were prospectively undertaken in 82 recipients before transplantation, at 6 months, and then at annual intervals for up to 10 years. Plaque in the internal carotid artery (ICA), external carotid artery, and common carotid artery was classified by type, location, extent, and degree of functional obstruction, and evaluated using multivariate analysis.Carotid plaque was present in 22.5% of patients at initial scanning, but increased to 56.6% by 7-10 years after transplantation, especially in the ICA and common carotid artery. Both the severity and extent of plaque increased, and plaque became more complex and heterogeneous with time after transplantation (P0.001). Carotid plaque was associated with older age, current cigarette smoking, hyperphosphatemia, hypoalbuminemia, duration of pretransplantation dialysis, and presence of lower limb plaque (P0.05-0.001). The severity of carotid plaque increased in older, hypertensive recipients and was associated with metabolic acidosis and hyperphosphatemia (all P0.05). Severity of ICA disease correlated with disease in the contralateral ICA (r=0.57, P0.001) and femoral arteries (r=0.42, P0.001). Paradoxically, each carotid artery progressed independently of the other. ICA disease severity progressed when heterogenous, calcified, or new plaque was present on scanning, and with reduced renal transplant function (P0.01-0.001). The mean ICA blood flow remained stable with time but was progressively impaired by hypertension, fasting hyperglycemia, and a lower prednisolone dose (P0.05). Cerebrovascular events occurred in only four patients and were unrelated to carotid disease, implying relative plaque stability.Extensive carotid vascular wall abnormalities increased significantly despite kidney and pancreas transplantation. Initiation of plaque was associated with systemic factors, whereas progression of established plaque was largely influenced by local factors within the arterial wall.

As more and more polymorphisms are discovered in the genes encoding cytokines, a crucial question is whether this polymorphism has any functional effect. One of the most widely studied cytokine genes in this respect is the gene encoding human TNF-alpha. Much of the literature investigating the issue of whether TNF-alpha promoter polymorphisms have any functional effect on TNF-alpha transcription or influence disease susceptibility appears to report negative results, giving the appearance and leading some authors to conclude that polymorphism at this locus is functionally silent and exists only because of linkage disequilibrium with selectable HLA alleles. This review presents a new analysis of the available data which suggests that polymorphism in the TNF-alpha promoter is not randomly distributed and therefore that it most likely does have some functional and selectable effect. Further, a comparison of available data suggests that there is more consensus in the literature than may at first appear to be the case.

The nucleotide sequence of a region downstream of the Listeria monocytogenes flagellin gene, flaA, revealed two putative chemotaxis genes, cheY and cheA. These genes have been shown to be transcribed as a bicistronic unit. In this study Tn916 delta E mutagenesis was used to generate two mutants, PF10 and PF16, which contain transposon inserts in the promoter region of this operon. These mutants were motile in liquid, but had reduced flagellin expression and were unable to burrow or swarm on soft agar plates. Complementation of the single transposon-copy mutant PF16 with cloned cheY and cheA in trans partially restored microaerotaxis and swarming on soft agar. The complemented strain did not exhibit any increase in flagellin production. Both PF10 and PF16 appear deficient in their ability to attach to the mouse fibroblast cell line 3T3.

Diabetic retinopathy (DR) is amenable to good diabetic control; however, only successful pancreas transplantation can achieve sustained normoglycaemia. The aim of this long-term study was to examine the course of DR in insulin-dependent diabetic recipients of a simultaneous kidney and pancreas transplant (SPK). Successful SPK recipients (n = 46) and failed pancreas transplant with a functioning kidney transplant (n = 8) were assessed by baseline and regular post-transplant ophthalmic examinations (n = 432) for up to 10 yr after SPK. At the time of SPK (n = 108 eyes), the mean duration of diabetes was 25 +/- 7 yr, ten eyes were blind, and 79% of eyes had advanced DR that had panretinal laser (panretinal photocoagulation, PRP. Successful SPK recipients had normal glucose control with a mean HBA1C of 5.2 +/- 0.6%. DR remained stable in 75% of both the study and control groups, with no difference between groups. The DR mostly evolved towards inactive proliferative DR. After SPK, 14% of non-blind eyes showed improvement of DR, 76% remained stable and 10% progressed. Early vitreous haemorrhage occurred in 6.1% of eyes, and was related to established DR. Cataract of all types increased after transplantation (p0.01), which reduced visual acuity (VA) in affected eyes. The mean overall VA remained unchanged for the study duration. In summary, uremic patients from diabetic nephropathy had a high prevalence of severe proliferative DR and blindness at the time of presentation for SPK. This was subsequently stabilised to inactive proliferative DR by appropriate laser therapy followed by metabolic control achieved by SPK.

Despite contemporary typing procedures for bone marrow transplantation (BMT), graft-versus-host disease (GVHD) continues to be a major complication of transplants performed between MHC-matched donors and recipients. Although GVHD can be alleviated by T cell depletion, this procedure increases the risk of graft failure and leukemic relapse and therefore is not a solution to the GVHD problem. The high degree of variation in the intensity of GVHD observed in different patients suggests that multiple non-MHC genetic factors influence GVHD severity. We hypothesize that, in addition to minor histocompatibility antigen disparities, polymorphisms in genes encoding immunologic effector molecules may be important factors influencing GVHD development. This study aims to explore this hypothesis by identifying non-MHC genes that influence the outcome of BMT in a murine model. In this model, B10.D2 donor leukocytes cause acute GVHD in (C57BL/6xDBA/2)F1 (B6D2F1) recipients, whereas DBA/2 donor leukocytes do not. To date, a locus on chromosome 1 has been identified as influencing the severity of GVHD in this model. Our current study shows that a locus on chromosome 2 acts independently of the chromosome 1 locus to also influence GVHD severity in this model. The region of chromosome 2 implicated in our study contains genes encoding beta2-microglobulin, the minor histocompatibility antigen H-3 and the pro-inflammatory cytokine IL-1.

Simultaneous pancreas and kidney transplantation (SPK) is capable of achieving normoglycemia, as assessed by oral glucose tolerance tests (OGTT), in the majority of diabetic recipients with end-stage renal disease. Despite its success over the last decade, standard ranges for OGTT after SPK are not available for comparison between studies and techniques. We examined 327 prospectively performed OGTT, undertaken in insulin-free bladder-drained SPK recipients, between 1 month and 10 yr after transplantation. Ranges of values for glucose, insulin and C-peptide were derived and areas under the time concentration curves calculated. The normal range of 2-h glucose values from SPK recipients was comparable to WHO criteria for the diagnosis of diabetes mellitus in a normal population; however, the fasting glucose fell below these levels. Fasting and area-under-the-curve (AUC) glucose were remarkably stable with time after SPK, although insulin fell with time after pancreas transplantation. These ranges may allow clinical evaluation of the endocrine function in individual SPK recipients, provide standards for comparison between different centers and techniques, and provide targets for islet transplantation.

While oral ganciclovir (OGCV) has been used as primary prophylaxis in patients at high risk for cytomegalovirus (CMV) infection after solid organ transplantation, its value in secondary prophylaxis is unknown. We have examined the use of OGCV as maintenance therapy following confirmed CMV infection on 16 occasions in 15 patients, in kidney (n = 4), kidney-pancreas (n = 3) lung (n = 6) and heart-lung (n = 1) recipients. OGCV was used in two distinct clinical situations. One group (n = 7) received OGCV as consolidation therapy (mean duration of 21 +/- 5 d) following i.v. ganciclovir induction therapy (mean duration of 16 +/- 5 d) for acute CMV infection, and sustained remission was achieved in 6/7 patients for a median follow-up of 300 d. In the second scenario, OGCV suppressed clinical disease in all patients with relapsing CMV infection (n = 9), with minimal or absent toxicity with a median follow-up of 152 d. OGCV reduced morbidity by allowing removal of central lines used for long-term i.v. therapy in patients with poor peripheral access. Hence, this study demonstrates the safe and effective use of OGCV as consolidation therapy after standard induction treatment with i.v. ganciclovir, and as long-term suppressive therapy in transplant recipients with recurrent CMV infection.

Current insulin therapies for control of glucose metabolism in patients with type I diabetes mellitus prevent major metabolic consequences of insulin deficiency, but none prevents or arrests long-term complications. In experimental models of canine diabetes, retinopathy, neuropathy, and nephropathy have been shown to develop within 5 years. The aim of this study was to determine in a canine model whether glucose control provided by segmental duct-occluded pancreas autografts could prevent the long-term complications of diabetes.Thirty-five outbred mongrel dogs underwent segmental pancreas autotransplantation with residual pancreatectomy. Follow-up over 5 years included endocrine, retinal fundus photography, fluorescein angiography, and nerve conduction studies. At endpoint, analysis of organ specific changes was undertaken.Long-term survival was achieved in 14 dogs for 4 to 5 years and in 3 dogs for 3 to 5 years. Glycosylated hemoglobin levels remained within normal limits, although response to glucose challenge was suboptimal. Fundus photography and fluorescein angiography demonstrated the absence of retinal vascular aneurisms, capillary leakage, and obliteration. Retinal digest showed no vascular changes and normal endothelial/pericyte ratios. Nerve conduction was normal, and histology of nerves revealed normal density of myelinated fibers and absence of intrafascicular vessels and glycogen deposits, with no change in spectrum of fiber diameters and ovoids. Renal histology revealed no evidence of nephropathy with normal glomerular basement membranes.We have demonstrated that duct-occluded segmental pancreatic autografts are capable of providing satisfactory metabolic control for up to 5 years, thereby preventing development of the long-term microvascular complications of diabetes.

A murine CTLA4/Fc gamma2a heavy chain (mCTLA4-Fc) chimeric fusion molecule was used in B6AF1 recipients of BALB/c pancreatic islet allografts to study the induction and maintenance of tolerance following inhibition of the CD28-B7 pathway for T cell activation. Donor-specific tolerance was achieved by administering 100 microg of mCTLA4-Fc on alternate days for 14 days (8 total doses) or a single 500 microg dose of mCTLA4-Fc on day 2 after transplant. Tolerance was mediated by long-lived peripheral lymphocytes and showed features of organ and alloantigen specificity. Whereas tolerance could not be established in allograft recipients receiving simultaneous mCTLA4-Fc and rIL-2, previously tolerant animals did not reject their grafts when given IL-2, suggesting that the induction and maintenance phases of tolerance were distinct and separate. The maintenance of donor-specific tolerance was an active immunologic process that was CD4+ T cell dependent and could be adoptively transferred to naive lymphocytes, but could not be explained by apoptosis or deletion of alloreactive T cells. Although an IL-2-sensitive mechanism such as anergy may contribute toward the induction of tolerance, its maintenance involves active suppression.

An Arabidopsis 14-3-3 protein, AFT1, was used as a 'bait' in the two-hybrid system to identify its interacting proteins. One of the candidate proteins, APX3, was identified as a putative peroxisomal membrane-bound ascorbate peroxidase. Ascorbate peroxidases are important defense enzymes that protect plant cells from oxidative stress damage. DNA blot analysis indicates that APX3 is encoded by a single-copy gene in the Arabidopsis genome. RNA blot analyses show that APX3 transcript levels increase slightly in response to cold, UV light, and treatments with hydrogen peroxide and paraquat. The activity of APX3 in Arabidopsis may be controlled in two ways: its enzymatic activity through protein-protein interactions and its transcription by transcriptional or posttranscriptional regulation.

The results of simultaneous pancreas and kidney transplantation (SPK) cannot be matched by pancreas transplantation alone (PTA), in part because an independent diagnosis of pancreas graft rejection remains difficult. The relationship between rejection of the pancreas and rejection of the kidney is poorly understood, and it is not known whether simultaneous transplantation of both organs confers true protection to either graft. To study these questions, reliable canine allotransplant models of kidney transplantation alone (KTA), PTA, and SPK were established. Sixty-seven mongrel dogs received KTA (n=21), PTA (n=23), or SPK (n=23) with either no immunosuppression, low-dose cyclosporine (CsA)-based immunosuppression, or high-dose CsA-based immunosuppression. Needle core biopsy (NCB) and fine needle aspiration biopsy (FNAB) were performed at 0, 2, 4, 7, 9, 11, 14, 21, and 30 days or at the time of graft failure. Pancreas and kidney graft survival after SPK was significantly shorter in dogs given low-dose CsA than in dogs given high-dose CsA (pancreas, P0.04; kidney, P0.03). Concurrent NCBs and FNABs were performed on 227 occasions in pancreas grafts and 229 occasions in kidney grafts. The time to initial evidence of rejection by NCB was not different in any immunosuppressed group. Synchronous rejection occurred in 73% of immunosuppressed SPK biopsies. Kidney-only rejection occurred in 23% of biopsies and pancreas-only rejection occurred in only 3% after SPK. All markers of pancreas graft rejection were poor, with the most sensitive being NCB of the simultaneously transplanted kidney. In summary, recipients of SPK required more immunosuppression than recipients of PTA, and improved PTA survival should be achievable with more sensitive markers of rejection. Markers of kidney rejection were the most sensitive indicators of pancreas rejection, and independent pancreas rejection was uncommon after SPK.

Techniques were used for viewing the true cytosolic surfaces of the membranes of intracellular organelles by field emission scanning electron microscopy (SEM). Cells of Paramecium multimicronucleatum were fed briefly with magnetic beads followed by a chase which advanced the newly formed digestive vacuoles (DVs) to predetermined ages. These bead-containing phagosomes were isolated from the whole cell homogenates with a magnet and were determined to be intact by fluorescence microscopy. Antigenically, these DVs were similar to those in situ. The DVs prepared for transmission electron microscopy or SEM showed extensive adherence of cellular debris. The use of 0.2 M KCl in the wash buffer eliminated much of this debris and exposed the true vacuolar surfaces. Three populations of tightly bound vesicles and numerous globular particles of 10 to 20 nm became visible on the DV surfaces. The attached vesicles, having diameters of approximately 300 and approximately 200 nm each, corresponded to the acidosomes and lysosomes that are known to be associated with the DV-I and DV-II, respectively. High resolution SEM also revealed a third set of small vesicles (50-150 nm), which were previously not known to be associated with DVs. The 10 to 20 nm globular particles were judged to be the cytosolic extensions of transmembrane protein complexes as their patterns of distribution on DVs of various ages corresponded to the transmembrane particles previously seen in these membranes in freeze-fracture studies.

In this canine study, glucose homeostasis after clinical pancreas transplantation is complex, with the relative effect of systemic versus portal delivery of insulin remaining unresolved. Thirty-two pancreatectomized dogs received either systemic venous drainage (SVD) with bladder exocrine drainage (n = 16), or portal venous drainage (PVD) with gastric exocrine drainage (n = 16). Cyclosporine (CsA) based immunosuppression was commenced on day -7. The effect of immunosuppression was a significant increase in fasting blood glucose (FBGL) (P = 0.002), fasting insulin (P = 0.024), AUC for insulin (P = 0.009), and K values decreased (P = 0.009). FBGL and K values remained abnormal after transplantation with no significant difference seen between SVD and PVD. However, fasting insulin became significantly lower after PVD and AUC insulin fell in both groups. CsA levels fell in both groups after transplantation, mirroring the fall in AUC insulin, and implicating CsA as a major cause of peripheral resistance to insulin. In conclusion, PVD did not demonstrate a significant advantage over SVD in handling an intravenous glucose challenge. The need for pancreatectomy in large animals may make them an unsatisfactory experimental model to evaluate the glucoregulatory effects of pancreas allotransplantation.

A model of sensitization by intraperitoneal lymph node inoculation was developed to test the hypothesis that hyperacute rejection (HAR) could occur in sensitized recipients of vascularized pancreas allografts. Ten pairs of outbred mongrel dogs that were lymphocytotoxic cross-match assay negative were inoculated with homogenized lymph nodes on either three or four occasions at fortnightly intervals before renal transplantation. A renal allograft from the same donor was used to test the HAR response and to further enhance sensitization by rejection of a vascularized organ. Pancreas transplants were performed 2 weeks later, with biopsies of the graft and blood samples taken at 0, 10, 20, and 30 min and then at 30-min intervals until the grafts were no longer viable. All renal and pancreas grafts were rejected in a classical hyperacute pattern. Within 4 min of revascularization of the pancreas, central lobular hemorrhage and vascular congestion appeared, followed by general edema. Histology demonstrated parallel changes of edema, vascular congestion, necrosis, hemorrhage, and leukocytic infiltrate, which all preceded graft infarction. A sharp decline in both arterial and venous white blood cell count and platelets occurred within 10 min of revascularization with initial sequestration and subsequent release of platelets from the graft (P=0.02). In summary, HAR of the allografted pancreas can be observed by the surgeon within minutes of revascularization, with predictable macroscopic and microscopic changes. This study supports the use of routine lymphocytotoxic cross-match tests for all recipients of pancreas transplants and implies that particular care is warranted in regraft pancreas allograft recipients.

Post-transplant erythrocytosis (PTE) has been increasingly recognized as a complication of kidney transplantation, and several risk factors have been defined. Recent evidence suggests renal function may also play a role in hematological recovery after transplantation and risk of PTE. In this study of kidney transplant recipients (n = 123), simultaneous Tc99m DTPA GFR (n = 710) and hemoglobin levels were compared with possible clinical determinants. The frequency histogram of post-transplant hemoglobin was bell-shaped and continuously distributed above and below the arbitrary definition of PTE, suggesting that PTE is not a separate disease entity. Hemoglobin reached a plateau at 12 months after transplantation and was correlated with isotopic GFR (r = 0.46, p0.001). This consistent and statistically independent relationship became prominent 3 months after transplantation. Hemoglobin was independently predicted by multivariate analysis by time after transplantation, presence of polycystic renal disease, greater serum albumin and reduced serum urea (which in turn were reflected by number of infective and rejection episodes), shorter kidney anastomosis time and a higher GFR, but not by immunosuppressive therapy. Rejection or infection episodes impaired hematological recovery. The independent determinants of GFR included hematological recovery. The independent determinants of GFR included hemoglobin level, kidneys from young, male donors, fewer HLA-DR mismatches and rejection episodes, shorter time on dialysis and greater azathioprine dose. Renal function was not altered by therapeutic phlebotomy. Determination of hemoglobin level by both donor and recipient variables supports the relevance of tubular and glomerular function in control of erythrocystosis after renal transplantation. A role for renin-angiotensin mediation in the alteration of intraglomerular hemodynamics and erythropoietin secretion is postulated.

Listeria monocytogenes and Listeria innocua differ markedly in virulence but are indistinguishable by classical taxonomic criteria. Both species are actively motile and produce abundant flagellin at 22 °C. We have found, however, noticeable differences between L. monocytogenes and L. innocua in motility and flagellin production at 37 °C. At this temperature, L. monocytogenes strains were virtually nonmotile and produced little or no detectable flagellin, whereas strains of L. innocua were frequently motile and produced substantial amounts of flagellin. This flagellin was recognized by a Listeria genus-specific monoclonal antibody that also recognized flagellin produced at 22 °C. These results suggest differential regulation of flagellin production between L. monocytogenes and L. innocua at 37 °C.Key words: Listeria monocytogenes, Listeria innocua, flagellin, motility, temperature, pathogenesis.

Impairment of glomerular filtration rate (GFR) after simultaneous pancreas and kidney (SPK) transplantation is an important marker of chronic renal rejection and recurrence of diabetic glomerulopathy. The use of unmodified serum creatinine to estimate GFR, however, is limited by variations in muscle mass. In this study, predictive factors for long-term GFR were evaluated in consecutive SPK recipients (n = 33) using a Tc99m DTPA GFR reference method between 90 days and 6 years after transplantation (n = 136 measurements). Substantial variability between serum creatinine and isotopic GFR after SPK (R2 = 0.30) high-lighted the inaccuracy of an unmodified serum creatinine in the evaluation of GFR. Factors which predicted GFR apart from serum creatinine included age, sex, height and body weight. A detailed formula was derived for accurate estimation of GFR (ml/min) = [71.4 (male) or 50.4 (female)] + 5520/creatinine (mumol/l) + 0.27 x body weight (kg) - 0.50 x age (yr) - 0.29 x height (cm). This formula was more accurate in estimation of GFR in SPK recipients than six published predictive methods which were derived from chronic renal failure patients using creatinine clearance. All of these methods overestimated GFR at lower levels of renal function. Most correlated poorly with Tc99m DTPA GFR and contained a generalized systematic overestimation of GFR which ranged from 4.7 to 8.4 ml/min (p0.05). A simplified version for rapid calculation was also derived as GFR (ml/min) = [25 (male) or 5 (female)] + 5000/creatinine (mumol/l).(ABSTRACT TRUNCATED AT 250 WORDS)

Although many cytokines have been previously implicated in graft-versus-host disease (GVHD), no study to date has comprehensively evaluated their expression over time or in different tissues affected by GVHD. Using a semi-quantitative reverse transcriptase-PCR technique and a murine model of acute GVHD, we have evaluated the expression levels of mRNA for a wide range of cytokines in spleen, gut and liver tissues at weekly intervals after bone marrow transfer. The earliest cytokine responses seen were increases in IL-2, IL-10, IFN-gamma, MIP-1 alpha and TNF-alpha in the spleen, suggesting a primarily Th1 pathway. Other cytokines (IL-1 alpha, IL-10 and MIP-1 alpha) were persistently elevated in GVHD mice, but were variable depending on the tissue. These data demonstrate that a wide range of cytokines are involved in the GVHD response and that their kinetic pattern of expression is different in various affected tissues.