Lyt-23+ cyclophosphamide-sensitive T cells regulate the activity of an interleukin 2 inhibitor in vivo.

Sera of thymus-bearing normal mice contain high levels of Interleukin 2 (II-2) inhibitor, whereas sera of athymic nu/nu mice do not. Evidence is presented that cyclophosphamide-sensitive Lyt-23+ T cells induce high II-2 inhibitor activity in the recipient nu/nu mice in the course of a graft-vs.-host reaction. The II-2 inhibitor has an approximately 50,000 mol wt. Its function is neither antigen specific nor H-2 restricted. During ontogeny, its activity parallels the development of T cell reactivity, i.e., it is absent both in the amniotic fluid and in sera of unborn mice, but increases to high levels during the early postnatal phase. The II-2 inhibitor described is viewed as an example of a T cell-dependent, in vivo regulatory mechanism able to effectively counteract the nonspecific activity of the Lyt-1+ helper T cell-derived II-2. Because the II-2 inhibitor activity is rather high in vivo, II-2 activity will exist only in close proximity to its producer cell, thereby maintaining specificity during the in vivo induction of cytotoxic T lymphocytes

Alloreactive and H-2-restricted Lyt 23 cytotoxic T lymphocytes derive from a common pool of antecedent Lyt 123 precursors.

If the collaborative requirement of Lyt 1 T helper cells is bypassed by the Lyt 1 T cell-derived mediator of T help, termed Il-2, upon antigenic stimulation, PNA+ Lyt 123 thymocytes differentiate into either alloreactive or H-2-restricted PNA- Lyt 23 cytotoxic effector cells. Along the differentiation pathway from Lyt 123 leads to 23 effector cells, cytolytic activity is carried out by T cells that still express the Lyt 123 phenotype. The data establish that Lyt 23 CTL are produced by differentiation from antecedent Lyt 123 cells.

Frequency analysis of cytotoxic T lymphocyte precursors in chimeric mice. Evidence for intrathymic maturation of clonally distinct self-major histocompatibility complex- and allo-major histocompatiblilty complex-restricted virus-specific T cells.

To study whether the thymic major histocompatibility complex (MHC) imposes a constraint on the receptor repertoire of maturating cytotoxic T lymphocyte (CTL) precursors, the restriction phenotypes of virus-specific CTL of MHC-compatible and of MHC-incompatible thymus- and bone marrow-grafted (A X B)F1 chimeric mice were compared. Dependent on the mode of in vitro sensitization, thymocytes or splenocytes of both types of chimeric mice generated Sendai virus-specific, self-MHC-or allo-MHC-restricted CTL. By applying the limiting-dilution technique, the CTL-precursor (CTL-P) frequencies of self-MHC-restricted and allo-MHC-restricted virus-specific T cells as well as of alloreactive T cells were determined. The data obtained revealed that independent of MHC differences between thymus and bone marrow, the frequencies of self-MHC-restricted and allo-MHC-restricted CTL-P were comparable, and in the same older of magnitude as those previously determined in conventionally reared mice. Self-MHC-restricted, virus-specific CTL-P were in a three- to fivefold excess over allo-MHC-restricted CTL-P. A segregation analysis revealed that clonally distinct CTL-P give rise to either self-restricted or allo-MHC-restricted, virus-specific CTL. Both sets were found not only in the spleen, but also in the thymus of chimeric mice, formally demonstrating the intrathymic differentiation pathway of self-MHC as well of allo-MHC-restricted CTL-P. These data reveal no major constraint of the thymic MHC on the capacity of T cells to recognize viral antigens either in the context of self-MHC or of allogeneic MHC products.

Dissection of the proliferative and differentiative signals controlling murine cytotoxic T lymphocyte responses.

Evidence is presented that interleukin 2 (IL-2) is not sufficient to cause the differentiation of primary cytotoxic T lymphocytes (CTL). Sources of IL-2 were compared for their ability to cause proliferation as well as differentiation into CTL. Whereas all factors caused proliferation, only the crude Con A supernatant had cytotoxic T cell differentiation factor (CTDF) activity. Furthermore, factors absorbed with an IL-2-dependent cell line to remove IL-2 still retained CTDF activity. Thus, IL-2 functions to cause clonal expansion of CTL precursors preactivated by antigen or mitogen, but for their differentiation into CTL, an additional factor is required, here called CTDF.

[Teaching non-technical skills for critical incidents: Crisis resource management training for medical students]. / Vermittlung von "soft skills" für Belastungssituationen: "Crisis-resource-management"--Kurs für Studierende.

BACKGROUND: Physicians have to demonstrate non-technical skills, such as communication and team leading skills, while coping with critical incidents. These skills are not taught during medical education. A crisis resource management (CRM) training was established for 4th to 6th year medical students using a full-scale simulator mannikin (Emergency Care Simulator, ECS, METI). PATIENTS AND METHODS: The learning objectives of the course were defined according to the key points of Gaba's CRM concept. The training consisted of theoretical and practical parts (3 simulation scenarios with debriefing). Students' self-assessment before and after the training provided the data for evaluation of the training outcome. RESULTS: A total of 65 students took part in the training. The course was well received in terms of overall course quality, debriefings and didactic presentation, the mean overall mark being 1.4 (1: best, 6: worst). After the course students felt significantly more confident when facing incidents in clinical practice. The main learning objectives were achieved. CONCLUSION: The effectiveness of applying the widely used ECS full-scale simulator in interdisciplinary teaching has been demonstrated. The training exposes students to crisis resource management issues and motivates them to develop non-technical skills.

Computational Network Analysis for Drug Toxicity Prediction.

The computational prediction of compound effects from molecular data is an important task in hazard and risk assessment and pivotal for judging the safety of any drug, chemical or cosmetic compound. In particular, the identification of such compound effects at the level of molecular interaction networks can be helpful for the construction of adverse outcome pathways (AOPs). AOPs emerged as a guiding concept for toxicity prediction, because of the inherent mechanistic information of such networks. In fact, integrating molecular interactions in transcriptome analysis and observing expression changes in closely interacting genes might allow identifying the key molecular initiating events of compound toxicity.In this work we describe a computational approach that is suitable for the identification of such network modules from transcriptomics data, which is the major molecular readout of toxicogenomics studies. The approach is composed of different tools (1) for primary data analysis, i.e., the biostatistical quantification of the gene expression changes, (2) for functional annotation and prioritization of genes using literature mining, as well as (3) for the construction of an interaction network that consists of interactions with high confidence and the identification of predictive modules from these networks. We describe the different steps of the approach and demonstrate its performance with public data on drugs that induce hepatic and cardiac toxicity.

ToxDB: pathway-level interpretation of drug-treatment data.

MOTIVATION: Extensive drug treatment gene expression data have been generated in order to identify biomarkers that are predictive for toxicity or to classify compounds. However, such patterns are often highly variable across compounds and lack robustness. We and others have previously shown that supervised expression patterns based on pathway concepts rather than unsupervised patterns are more robust and can be used to assess toxicity for entire classes of drugs more reliably. RESULTS: We have developed a database, ToxDB, for the analysis of the functional consequences of drug treatment at the pathway level. We have collected 2694 pathway concepts and computed numerical response scores of these pathways for 437 drugs and chemicals and 7464 different experimental conditions. ToxDB provides functionalities for exploring these pathway responses by offering tools for visualization and differential analysis allowing for comparisons of different treatment parameters and for linking this data with toxicity annotation and chemical information.Database URL:http://toxdb.molgen.mpg.de.

Heterozygous expansion of the GAA tract of the X25/frataxin gene is associated with insulin resistance in humans.

Friedreich's ataxia (FA) is an autosomal recessive disease that has been attributed to a GAA triplet repeat expansion in the first intron of the X25/frataxin gene. Impaired glucose tolerance is present in up to 39% of FA patients, and clinically apparent diabetes is seen in approximately 18% of the affected individuals. Subjects carrying the X25/frataxin GAA repeat in a heterozygous state do not develop FA and, therefore, represent an ideal model to study the underlying metabolic defects that contribute to the diabetes associated with this disorder. In the present study, we have compared 11 first-degree relatives of FA patients (i.e., parents or heterozygous siblings of FA patients) with matched normal control subjects to study the parameters of glucose metabolism. An oral glucose tolerance test revealed diabetes in one of the heterozygous subjects who was excluded from further analyses. Using an octreotide-based quantification of insulin sensitivity, 8 of the remaining 10 study subjects showed pronounced insulin resistance, reflecting a significant difference from the control group (P = 0.001). In conclusion, a heterozygous expansion of the X25/frataxin GAA repeat in healthy individuals is associated with insulin resistance and might be considered a genetic co-factor in the pathogenesis of mitochondrial subtypes of diabetes.

The GAA repeat expansion in intron 1 of the frataxin gene is related to the severity of cardiac manifestation in patients with Friedreich's ataxia.

Friedreich's ataxia is an autosomal recessive disorder characterized by spinocerebellar degeneration. It is caused by an unstable GAA trinucleotide repeat expansion (>120 repeats) in the first intron of the frataxin gene on chromosome 9 (9q13) in both alleles. Concentric left ventricular hypertrophy has been recognized as the major cardiac manifestation of Friedreich's ataxia. Our aim was to investigate the influence of the frataxin repeat length on cardiac hypertrophy in patients with Friedreich's ataxia and in patients with hypertrophic and dilated cardiomyopathy. Thirty-one patients with Friedreich's ataxia, 86 patients with hypertrophic cardiomyopathy, 134 patients with dilated cardiomyopathy, and 32 healthy individuals without cardiac disease were analysed by electrocardiography and 2D-M-mode echocardiography. Then, the size of the frataxin repeat was determined by polymerase chain reaction (PCR) and agarose gel electrophoresis. The number of GAA repeats in patients with hypertrophic and dilated cardiomyopathy was not different from the length in patients without cardiac disease (hypertrophic cardiomyopathy, 8+/-2 repeats on GAA 1 allele and 11+/-5 repeats on GAA 2 allele; dilated cardiomyopathy, 7+/-2 repeats on GAA 1 allele and 11+/-5 repeats on GAA 2 allele; Control, 9+/-1 repeats on GAA 1 allele and 12+/-6 repeats on GAA 2 allele). The septal and posterior wall thickness of these patients was not related to the GAA repeat length. All patients with Friedreich's ataxia had two enlarged alleles with a mean GAA repeat length of 757+/-316 and 1012+/-231, respectively. The lengths of both alleles were significantly greater than the lengths in the controls (P<0.0001), patients with hypertrophic cardiomyopathy (P<0.0001) and dilated cardiomyopathy (P<0.0001). A significant correlation was revealed between interventricular septal hypertrophy and frataxin repeat length in the smaller allele. Furthermore, the ratio of septal to posterior wall thickness was significantly correlated to GAA repeat size on the smaller allele. In conclusion, the size of the GAA repeat on the smaller allele in the frataxin gene is associated with the degree of left ventricular hypertrophy in patients with Friedreich's ataxia but is not related to the severity of hypertrophic cardiomyopathy.

A complex containing at least one zinc dependent HeLa nuclear protein binds to the intronic (gaa)(n) block of the frataxin gene.

We analyzed HeLa nuclear proteins binding to the (gaa)(n) harbouring intron 1 of nine frataxin alleles and characterized the structures of the repeats. Fragments with blocks longer than (gaa)(9) form spontaneously different intramolecular H-y topoisomeres in linear state. The observed triplexes depend on the length of the repeat. Interruption of the perfectly repeated (gaa)(n) block entails two structural regions. At least two HeLa nuclear proteins bind to the (gaa)(n) fragments resulting in a distinct major retarded complex as revealed by EMSA. One of these proteins is zinc dependent. Importantly, the fragment harbouring (gan)(121) binds additional proteins. Protein binding appears to be locus specific, and the binding affinity was found to be not random. The affinities of the different target fragments varied by a factor of four. Binding affinities of the fragments were not obviously correlated to differences in the composition of the repeats. DNase I footprinting revealed only weakly protected binding regions, but multiple HS sites in the repeat regions of the fragments. These findings and the fact, that DNA conformers observed in EMSA and electron microscopical experiments bind proteins, lead to the assumption that the proteins recognize, both, B-DNA and triple helical structures, but with different affinity. Possible functions of the proteins are discussed in the context of transformation of triple helical structures into B-DNA and the pathogenesis of FRDA.

Extrapyramidal motor signs in degenerative ataxias.

BACKGROUND: Extrapyramidal motor signs (EPS) are well-known symptoms of degenerative ataxia. However, little is known about frequency and appearance of EPS in subtypes of ataxia. METHODS: We characterized 311 patients with ataxia clinically and genetically. Course of the disease and EPS were investigated according to a standardized protocol. Diagnostic and prognostic impact of EPS in subtypes of ataxia was analyzed by Kaplan-Meier plots. RESULTS: Extrapyramidal motor signs occurred in all forms of ataxia, but frequency and type of EPS varied between genetically and clinically defined subtypes. Postural tremor in hereditary ataxias was typical for spinocerebellar ataxia type 2 (SCA2). Dystonia was generally rare in ataxias, but, if present, suggested SCA3. We observed a parkinsonian variant of SCA3 in which parkinsonism was present in the beginning of the disease and responded well to levodopa therapy, leading to diagnostic confusion. Parkinsonism in SCA3 was independent of CAG repeat length but ran in families, suggesting modifying genes. In idiopathic sporadic cerebellar ataxia (ISCA), EPS are more frequent in late-onset than in early-onset forms. In 50% of ISCA patients with parkinsonism, the diagnosis of multiple system atrophy remained questionable because of normal autonomic function. CONCLUSIONS: Extrapyramidal motor signs can help to predict the genetic subtype of ataxia. Extrapyramidal motor signs were more frequent in genetic subtypes in which basal ganglia affection has been demonstrated by postmortem studies. However, no type of EPS was specific for an underlying mutation. In ISCA, EPS are an adverse prognostic factor. Parkinsonism is especially associated with a more rapid course of the disease. Arch Neurol. 2000;57:1495-1500

Oxidative stress in patients with Friedreich ataxia.

Increased generation of reactive oxygen species may underlie the pathophysiology of Friedreich ataxia (FRDA). The authors measured concentrations of 8-hydroxy-2'-deoxyguanosine (8OH2'dG), a marker of oxidative DNA damage, in urine and of dihydroxybenzoic acid (DHBA), a marker of hydroxyl radical attack, in plasma of 33 patients with FRDA. They found a 2.6-fold increase in normalized urinary 8OH2'dG but no change in plasma DHBA as compared with controls. Oral treatment with 5 mg/kg/day of the antioxidant idebenone for 8 weeks significantly decreased urinary 8OH2'dG concentrations, indicating that 8OH2'dG may be useful in monitoring therapeutic interventions in patients with FRDA.-1721

A rapid colorimetric assay for the determination of IL-2-producing helper T cell frequencies.

Interleukin 2 (IL-2) activity is tested in conditioned media by assessing its ability to support proliferation of selected IL-2 dependent T cell lines, conventionally measured by [3H]thymidine incorporation. Here, we compare this [3H]thymidine uptake test for measuring IL-2 activity with a rapid and sensitive colorimetric method which is based on the ability of viable cells to cleave 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). The sensitivity of the colorimetric method was dependent on the indicator cell line used, being greatest with the cytotoxic T cell line 16 (CTLL-16). The colorimetric method is at least as sensitive as [3H]thymidine uptake tests, does not rely on radioactivity, and is ideally suited to screen large numbers of individual samples for IL-2 activity. The latter point was demonstrated by calculating IL-2-producing helper T cell frequencies in heterogeneous murine lymphocyte populations: in this assay, splenic T cells were clonally expanded under limiting dilution conditions and supernatants conditioned by these in vitro growing T cell clones were tested for IL-2 activity with the colorimetric method. This allowed us to obtain reliable estimates of the frequency of progenitor cells of IL-2-producing T cell clones in various populations.

Mitochondrial impairment of human muscle in Friedreich ataxia in vivo.

Friedreich ataxia occurs due to mutations in the gene encoding the mitochondrial protein frataxin. This (31)P magnetic resonance spectroscopy study on the calf muscle of Friedreich ataxia patients provides in vivo evidence of a severe impairment of mitochondrial function. Mitochondrial adenosine triphosphate resynthesis was studied by means of the post-exercise recovery of phosphocreatine. After ischemic exercise in calf muscles of all patients, phosphocreatine recovery was dramatically delayed. Time constants of recovery correlated with mutations of the frataxin gene, the age of the patients, and disease duration. (31)P magnetic resonance spectroscopy represents the first expedient tool for monitoring therapeutic trials in Friedreich ataxia non-invasively.

The in vivo effects of interleukin 2 (TCGF).

This brief review of our experiments concerning the in vivo activity of crude Il-2 led us to the following conclusion: The first is the existence, in vivo, of a cyclophosphamide-sensitive T-cell controlling the activity of a serum born Il-2 inhibitor in thymus-bearing normal mice. Under in vivo conditions which are characterized by high Il-2 inhibitor activities, locally applied Il-2 administered along with antigen amplified in vivo CTL-responsiveness, yet the effect observed was poor. Crude Il-2 proved to be a potent immuno-enhancing agent in the athymic (nu/nu) mouse, which lacks Il-2 inhibitor activity. It was found that together with antigen administration of Il-2 to nude mice results in the generation of highly reactive T-helper cells, as well as in the generation of alloreactive CTL.

Cross-linking of T-cell receptors is insufficient to induce IL-2 responsiveness (activation) in resting Lyt-2+ T cells. IL-4 or RIF are essential as second signal.

High-density (resting) murine Lyt-2+ T cells exposed in vitro to the ligand concanavalin A (Con A), or immobilized F23.1 monoclonal antibody (mAb) recognizing an allotypic determinant on the T-cell receptor (TCR), or high-density (resting) allogeneic B stimulator cells remain IL-2-unresponsive; such cells do not express functional IL-2 receptors unless reconstituted with accessory cells. We conclude that cross-linking of TCR is insufficient as signal to induce IL-2 responsiveness, that is, activation. Both the macrophage product RIF and the T-cell product interleukin-4 efficiently induce the IL-2 responsiveness in resting Lyt-2+ T cells exposed in vitro either to the ligand Con A, or to immobilized F23 mAb, or to nonimmunogenic allogeneic stimulator cells. We conclude that two restricting points control the induction of IL-2 responsiveness (activation) in antigen-driven Lyt-2+ T-cell responses, that is, cross-linking of TCR by way of presented antigen and "costimulator" activity expressed by accessory cells. Both RIF and IL-4 express costimulator activity, therefore replacing the requirement for accessory cells.

Sequence-based typing reveals a novel DLA-88 allele, DLA-88*04501, in a beagle family.

The dog is an important animal model for solid organ as well as stem cell allo transplantation. Methods such as cellular and serological typing and more recently sequence-based typing (SBT) have been used to discriminate tissue antigen disparity of donor and recipient. We applied SBT for the canine class I (DLA-88) and class II (DLA-DRB1) genes in beagle families prior stem cell transplantation. A novel DLA-88 (DLA-88*04501) allele in combination with a DLA-DRB1*01901 allele was found. Sequence comparison of exons 2 and 3 of the novel allele revealed most sequence identity to the DLA-88*01301 allele (96.15% identity at the nucleotide and 90.65% identity at the protein level).

Identification of a new HLA-B allele, HLA-B*4443, in a German family.

A novel human leukocyte antigen (HLA)-B allele is described. The allele was identified in a German blood donor of Caucasian origin. Because high-resolution HLA-typing using sequence-specific primers gave inconclusive results, sequence-based typing was performed. Nucleotide sequences of exons 2 and 3 most closely match with HLA-B*4417 and HLA-B*440301 (99.5% identity). The predicted protein sequence revealed a single amino acid substitution (D156L) compared with the HLA-B*4417 allele but two substitutions (Y113H, D116S) compared with the HLA-B*440301 allele. Therefore, the novel allele has been officially assigned HLA-B*4443 by the WHO Nomenclature Committee. The HLA-B*4443 allele was found with the A*2301, Cw*0401, B*4443, DRB1*0701, DRB4*0107, and DQB1*0202 haplotype.