An integrative approach to CTL epitope prediction: a combined algorithm integrating MHC class I binding, TAP transport efficiency, and proteasomal cleavage predictions

Reverse immunogenetic approaches attempt to optimize the selection of candidate epitopes, and thus minimize the experimental effort needed to identify new epitopes. When predicting cytotoxic T cell epitopes, the main focus has been on the highly specific MHC class I binding event. Methods have also been developed for predicting the antigen-processing steps preceding MHC class I binding, including proteasomal cleavage and transporter associated with antigen processing (TAP) transport efficiency. Here, we use a dataset obtained from the SYFPEITHI database to show that a method integrating predictions of MHC class I binding affinity, TAP transport efficiency, and C-terminal proteasomal cleavage outperforms any of the individual methods. Using an independent evaluation dataset of HIV epitopes from the Los Alamos database, the validity of the integrated method is confirmed. The performance of the integrated method is found to be significantly higher than that of the two publicly available prediction methods BIMAS and SYFPEITHI. To identify 85% of the epitopes in the HIV dataset, 9% and 10% of all possible nonamers in the HIV proteins must be tested when using the BIMAS and SYFPEITHI methods, respectively, for the selection of candidate epitopes. This number is reduced to 7% when using the integrated method. In practical terms, this means that the experimental effort needed to identify an epitope in a hypothetical protein with 85% probability is reduced by 20-30% when using the integrated method.The method is available at http://www.cbs.dtu.dk/services/NetCTL. Supplementary material is available at http://www.cbs.dtu.dk/suppl/immunology/CTL.php.     

Aspartate-like and glutamate-like immunoreactivities in the inferior olive and climbing fibre system: a light microscopic and semiquantitative electron microscopic study in rat and baboon (Papio anubis)

A post-embedding immunogold procedure was used to analyse, in a semiquantitative manner, the distributions of aspartate-like and glutamate-like immunoreactivities in the inferior olive and climbing fibre system in rats and baboons. The neurons in the inferior olive were uniformly labelled for aspartate as well as glutamate, indicating a 100% co-localization of these two amino acids in the cell bodies. The level of glutamate-like immunoreactivity in the climbing fibre terminals was similar to that in the parent cell bodies, as judged by a computer-assisted calculation of gold particle densities. In contrast, the level of aspartate-like immunoreactivity in the climbing fibre terminals was only one-seventh of that of the olivary neurons. No differences were found between the hemispheres and vermis. Nerve terminals in the inferior olive were generally moderately labelled with the aspartate antiserum, as were cell bodies of astrocytes. With a few exceptions, the results obtained in baboons were similar to those in rats. Notably, no evidence was found of an enrichment of aspartate-like immunoreactivity in climbing fibres. The present results do not support previous data suggesting that aspartate is the transmitter of the climbing fibres but indicate that glutamate or another excitatory compound should be considered as candidate for this role. Our findings show that the presence of aspartate-like immunoreactivity in cell bodies is an unreliable indicator of transmitter identity.     

Requests for euthanasia or physician-assisted suicide from older persons who do not have a severe disease: an interview study

OBJECTIVE: To determine how often requests are made for euthanasia and physician-assisted suicide (EAS) in the absence of severe disease and how such requests are dealt with in medical practice in The Netherlands. METHOD: Retrospective interview study. Participants: 125 general practitioners (GPs), 77 nursing home physicians (NHPs), and 208 clinical specialists. RESULTS: In The Netherlands, each year approximately 400 people request EAS, because they are 'weary of life'. Thirty per cent of all physicians have at some time received an explicit request for EAS in the absence of severe disease; 3% of all physicians had granted a request for EAS in such a case. Most requests for EAS to GPs in the absence of severe disease (n = 29) were made by single people aged 80 years and over. While their problems were most frequently of a social nature, 79% had one or more non-severe illnesses. Most GPs refused the request; half of them proposed an alternative treatment, which the patient often refused. Nineteen people who did not receive any treatment persisted in their wish to die; the request for EAS from 5 out of 10 patients who received one or more types of treatment was withdrawn or became less explicit. CONCLUSIONS: Most physicians in The Netherlands refuse requests for EAS in the absence of severe disease. Most patients persist in their request. In an ageing population more research is needed to provide physicians with practical interventions to prevent suicide and to make life bearable and satisfactory for elderly people who wish to die.     

The medullary projection from the mesencephalic trigeminal nucleus. An experimental study with comments on the intrinsic trigeminal connections

Summary The medullary projection from the mesencephalic trigeminal nucleus has been studied in cats where the wheat germ agglutinin-horseradish peroxidase complex has been used as a retrograde and anterograde tracer. All injections were made at the level of the caudal pole of the inferior olive and show that it is especially the lateral part of nucleus parvicellularis of the reticular formation which is the main area for termination of the fibres. In addition, it can not be excluded that the descending fibres also reach the medialmost part of the spinal trigeminal sensory nucleus (pars magnocellularis and the adjoining pars gelatinosa). All three cell types of the mesencephalic trigeminal nucleus are labelled, the large globular, the small globular and the polygonal cells. Some of these cells are only faintly labelled. The observations confirm previous studies of the intrinsic trigeminal connections, but show that when the wheat germ agglutinin-horseradish peroxidase complex is used as a tracer, the origin as well as the termination for the intrinsic fibres (also the commissural) can be studied in the same section. The use of this sensitive tracer also shows new details within the intrinsic connections. The findings are discussed with special reference to the recent observations by Ruggiero et al. (1982). These authors found that in rabbit and rat there is a more restricted termination for the descending mesencephalic trigeminal fibres. The discrepancies between the observations seem to indicate that there are species differences as regards the connections between the mesencephalic trigeminal nucleus and the nuclei in the lower part of the brain stem.     

Numerical Simulation and Experimental Validation of Blood Flow in Arteries with Structured-Tree Outflow Conditions

Blood flow in the large systemic arteries is modeled using one-dimensional equations derived from the axisymmetric Navier–Stokes equations for flow in compliant and tapering vessels. The arterial tree is truncated after the first few generations of large arteries with the remaining small arteries and arterioles providing outflow boundary conditions for the large arteries. By modeling the small arteries and arterioles as a structured tree, a semi-analytical approach based on a linearized version of the governing equations can be used to derive an expression for the root impedance of the structured tree in the frequency domain. In the time domain, this provides the proper outflow boundary condition. The structured tree is a binary asymmetric tree in which the radii of the daughter vessels are scaled linearly with the radius of the parent vessel. Blood flow and pressure in the large vessels are computed as functions of time and axial distance within each of the arteries. Comparison between the simulations and magnetic resonance measurements in the ascending aorta and nine peripheral locations in one individual shows excellent agreement between the two. © 2000 Biomedical Engineering Society. PAC00: 8719Uv     

Estimation of theophylline clearance during intravenous aminophylline infusions

The utility of predicting theophylline clearance (CL) from two serum concentrations obtained during continuous intravenous aminophylline infusion was examined in 16 stable, adult patients. Blood for theophylline measurement was obtained 0, 6, and 12 h after starting infusions and, thereafter, at 12-h intervals. EMIT was used to assay samples in multiple runs as they were obtained. Later, each sample was reassayed by EMIT within a single run. Bayesian least-squares regression and the algebraic method of Chiou were used to predict CL using the 0,6 and 0,12 h concentrations. "Actual" CL was measured by nonlinear least-squares regression of all concentrations obtained during prolonged infusions. Prediction bias and precision were assessed by calculating mean percent error (PCE) and mean absolute percent error (APCE), respectively. A three-way repeated-measures ANOVA was used to examine the effect of the method of CL prediction, assay procedure, and time interval between samples on PCE and APCE. Bayesian predictions were less biased and slightly more precise than Chiou predictions. The assay procedure had no effect on bias but precision was improved using a single-assay run. Predictions were less biased and more precise with 0,12 h versus 0,6 h data. Serum samples for theophylline measurement should be obtained after initiating constant intravenous aminophylline and again 8-12 h later in stable, adult patients. Prediction of CL with either of the concentration-based methods studied will then allow safe and rapid adjustment of dosage to achieve therapeutic serum concentrations.     

Hypercalcemia After Cosmetic Oil Injections: Unraveling Etiology,Pathogenesis, and Severity

Intramuscular injections of paraffin oil can cause foreign body granuloma formation and hypercalcemia. Macrophages with the ability to produce high levels of 1,25(OH)₂D₃ may induce the mineral disturbance, but no major series of patients have been published to date. Here, medical history, physical evaluation, biochemical, and urinary analysis for calcium homeostasis were obtained from 88 males, who 6 years previously had injected paraffin or synthol oil into skeletal muscle. Moreover, granuloma tissue from three men was cultured for 48 hours ex vivo to determine 1,25(OH)₂D₃ production supported by qPCR and immunohistochemistry of vitamin D metabolism and immune cell populations after treatment with 14 different drugs. The 88 men were stratified into men with hypercalcemia (34%), whereas normocalcemic men were separated into men with either normal (42%) or suppressed parathyroid hormone (PTH) (24%). All men had high calcium excretion, and nephrolithiasis was found in 48% of hypercalcemic men, 22% of normocalcemic men with normal PTH, and 47% of normocalcemic men with suppressed PTH. Risk factors for developing hypercalcemia were oil volume injected, injection of heated oil, high serum interleukin-2 receptor levels, and high urine calcium. High 1,25(OH)₂D₃/25OHD ratio, calcium excretion, and low PTH was associated with nephrolithiasis. The vitamin D activating enzyme CYP27B1 was markedly expressed in granuloma tissue, and 1,25(OH)₂D₃ was released in concentrations corresponding to 40% to 50% of the production by human kidney specimens. Dexamethasone, ketoconazole, and ciclosporin significantly suppressed granulomatous production of 1,25(OH)₂D₃. In conclusion, this study shows that injection of large oil volumes alters calcium homeostasis and increases the risk of nephrolithiasis. Hypercalciuria is an early sign of disease, and high granulomatous 1,25(OH)₂D₃ production is part of the cause. Prospective clinical trials are needed to determine if ciclosporin, ketoconazole, or other drugs can be used as prednisolone-sparing treatment. © 2020 American Society for Bone and Mineral Research (ASBMR).     

Applicability of yeast genetics to neurologic disease

As advances in gene mapping technology reveal genes associated with neurologic diseases, the need to identify a gene's normal function arises often. Experimental genetics is very useful in identifying a gene's function. It relies on model organisms both because it is not appropriate in humans, and because many processes are remarkably similar among eukaryotes. Many cellular processes have evolved once, and species differences are variations on a theme. Molecular genetic tools available in the yeast Saccharomyces cerevisiae provide a means to more rapidly reach an understanding of gene function, yielding substantial insight into the same process in humans. Yeast will never complain of headache or "spells," but do have expansions of trinucleotide repeats, prions, and other processes very much analogous to those underlying many neurologic diseases. In spite of the absence of a nervous system in yeast, yeast genetics has contributed substantial insight into neurologic diseases mechanisms. The real strength of yeast in studying human disease is in genetic analysis of gene function and in providing genetically powerful functional assays. Arch Neurol. 2000;57:1129-1134