Characterization of the dominant autoreactive T-cell epitope in spontaneous autoimmune haemolytic anaemia of the NZB mouse

NZB mice spontaneously develop autoimmune haemolytic anaemia (AIHA) due to a T helper-dependent autoantibody response against the erythrocyte anion channel protein, Band 3. Here, we characterize the recognition of the Band 3 sequence 861-874, which carries the dominant, I-E(d)-restricted T cell epitope. The ability of N and C-terminal truncated versions of peptide 861-874 to elicit NZB splenic T-cell proliferation indicated that the core epitope spans residues 862-870. Next, a set of alanine substitution analogues was tested to determine which residues functioned either as MHC anchor or TCR contact residues. A combination of proliferation and MHC:peptide binding assays identified residues 862(L), 864(V), 865(L), and 869(K) as I-E(d) anchor residues, and 868(V) as the only TCR contact residue. The ability of the wild-type sequence 861-874 to compete with a high affinity reference peptide for binding to I-E(d) indicates that the escape of pathogenic NZB T cells from purging of the autoreactive repertoire cannot be attributed to ineffective presentation of peptide 861-874 by its restricting element. It will now be possible to design altered peptide ligands of Band 3 861-874, in order to further dissect the mechanisms responsible for the maintenance and loss of T cell tolerance to RBC autoantigens, and to modulate the immune response in AIHA.     

Cross-presentation: underlying mechanisms and role in immune surveillance

Summary: It was originally thought that a cell's major histocompatibility complex (MHC) class I molecules presented peptides derived exclusively from proteins synthesized by the cell itself. However, in some circumstances, antigens from the extracellular environment can be presented on MHC class I molecules and stimulate CD8⁺ T‐cell immunity, a process termed cross‐presentation. Cross‐presentation was originally discovered as an obscure phenomenon in transplantation immunity. However, it is now clear that it is a major mechanism by which the immune system monitors tissues and phagocytes for the presence of foreign antigen. Cross‐presentation is the only pathway by which the immune system can detect and respond to viral infections or mutations that exclusively occur in parenchymal cells rather than in bone marrow‐derived antigen‐presenting cells (APCs). Professional APCs, such as dendritic cells, are the principal cells endowed with the capacity to cross‐present antigens. In this process, the APCs acquire proteins from other tissue cells through endocytic mechanisms, especially phagocytosis or macropinocytosis. The internalized antigen can then be processed through at least two different mechanisms. In one pathway, the antigen is transferred from the phagosome into the cytosol, where it is hydrolyzed by proteasomes into oligopeptides that are then transported by the transporter associated with antigen processing to MHC class I molecules in the endoplasmic reticulum or phagosomes. In a second pathway, the antigen is cleaved into peptides by endosomal proteases, particularly cathepsin S, and bound by class I molecules probably in the endocytic compartment itself. Depending on the nature of the antigen, one or both of these pathways can contribute to cross‐presentation in vivo. The outcome of cross‐presentation can be either tolerance or immunity. Which of these outcomes occurs is thought to depend on whether antigens are acquired by themselves alone, leading to tolerance, or with immunostimulatory signals, leading to immunity. One source of such signals is from dying cells that release immunostimulatory ‘danger’ signals that promote the generation of immunity to their cellular antigens. In addition to the critical role of cross‐presentation in normal immune physiology, this pathway has considerable potential for being exploited for developing subunit vaccines that elicit both CD4⁺ and CD8⁺ T‐cell immunity.     

Genome-wide examination of myoblast cell cycle withdrawal during differentiation.

Skeletal and cardiac myocytes cease division within weeks of birth. Although skeletal muscle retains limited capacity for regeneration through recruitment of satellite cells, resident populations of adult myocardial stem cells have not been identified. Because cell cycle withdrawal accompanies myocyte differentiation, we hypothesized that C2C12 cells, a mouse myoblast cell line previously used to characterize myocyte differentiation, also would provide a model for studying cell cycle withdrawal during differentiation. C2C12 cells were differentiated in culture medium containing horse serum and harvested at various time points to characterize the expression profiles of known cell cycle and myogenic regulatory factors by immunoblot analysis. BrdU incorporation decreased dramatically in confluent cultures 48 hr after addition of horse serum, as cells started to form myotubes. This finding was preceded by up-regulation of MyoD, followed by myogenin, and activation of Bcl-2. Cyclin D1 was expressed in proliferating cultures and became undetectable in cultures containing 40% fused myotubes, as levels of p21(WAF1/Cip1) increased and alpha-actin became detectable. Because C2C12 myoblasts withdraw from the cell cycle during myocyte differentiation following a course that recapitulates this process in vivo, we performed a genome-wide screen to identify other gene products involved in this process. Using microarrays containing approximately 10,000 minimally redundant mouse sequences that map to the UniGene database of the National Center for Biotechnology Information, we compared gene expression profiles between proliferating, differentiating, and differentiated C2C12 cells and verified candidate genes demonstrating differential expression by RT-PCR. Cluster analysis of differentially expressed genes revealed groups of gene products involved in cell cycle withdrawal, muscle differentiation, and apoptosis. In addition, we identified several genes, including DDAH2 and Ly-6A, whose expression specifically was up-regulated during cell cycle withdrawal coincident with early myoblast differentiation.     

Primary giant cell malignant fibrous histiocytoma of the kidney with staghorn calculi

Malignant fibrous histiocytomas (MFH) as primary renal tumours are rare, with less than 50 cases described in the literature. We report a case of primary renal MFH of giant cell type in a 56-year-old man, who presented with bilateral dull flank pain, intermittent gross haematuria and body weight loss (6 kg in 3 months). Intravenous urography, computerized tomography (CT) and magnetic resonance image (MRI) showed right ureteral stones with mild hydronephrosis, and a solid mass at the lower pole of the left kidney associated with staghorn calculi, as well as tumour thrombi in the left renal vein and inferior vena cava. Left radical nephrectomy and evacuation of tumour thrombi from the left renal vein and inferior vena cava were performed. Histopathologic examination revealed malignant fibrous histiocytoma (MFH) of giant cell type. To the best of our knowledge, this is the first report of primary renal MFH associated with staghorn calculi.     

Preantral follicle culture as a novel in vitro assay in reproductive toxicology testing in mammalian oocytes

The most common genetic disorder in humans, trisomy, is caused predominantly by errors in chromosome segregation during oogenesis. Isolated mouse oocytes resuming meiosis and progressing to metaphase II in vitro have recently been used to assess targets, aneugenic potential and sensitivity of oocytes to chemical exposures. In order to extend in vitro maturation tests to earlier stages of oogenesis, an in vitro assay with mouse preantral follicle cultures has been established. It permits the identification of direct and also indirect effects of environmental chemicals on the somatic compartment, the follicle and theca cells, that may lead to disturbances of oocyte growth, maturation and chromosome segregation. Early preantral follicles from prepubertal female mice are cultured in microdroplets for 12 days under strictly controlled conditions. The follicle-enclosed oocytes resume maturation, develop to metaphase II and become in vitro ovulated within 16 h after a physiological ovulatory stimulus with recombinant human gonadotrophins and epidermal growth factor. These oocytes grown and matured in vitro possess normal barrel-shaped spindles with well-aligned chromosomes. Their chromosomes segregate with high fidelity during anaphase I. The model aneugen colchicine induced a meiotic arrest and aneuploidy in these in vitro grown, follicle-enclosed oocytes in a dose-dependent manner, comparable to in vivo tests. Therefore, preantral follicle culture appears to provide an effective and reliable method to assess the influences of environmental mutagens, pharmaceutical agents and potentially endocrine disrupting chemicals on the fidelity of female meiosis.     

MPTP metabolites inhibit rat brain glutathione S-transferases

1-Methyl-4-phenyl-2,3-dihydropyridinium and 1-methyl-4-phenyl-pyridinium species, metabolites of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, non-competitively inhibit glutathione S-transferases of rat brain in vitro. The Ki values for 1-methyl-4-phenyl-2,3-dihydropyridinium bromide and 1-methyl-4-phenyl-pyridinium bromide are 0.67 and 0.3 mM, respectively. Inhibition of these enzymes may lead to impairment of cellular defense mechanisms.     

Genetic linkage maps of the West African clawed frog Xenopus tropicalis.

Amphibians, and particularly the African clawed frog Xenopus laevis, have been used for more than a century as models of vertebrate embryonic development. However, in many cases, elucidation of developmental functions of specific gene sequences could be severely impeded, because X. laevis is a tetraploid species, with multiple functional copies of many genes of interest. Recent studies have shifted focus to the West African or tropical clawed frog, X. tropicalis, the only known diploid species of the genus Xenopus. Here, we present two preliminary linkage maps, constructed by analysis of joint segregation of amplified fragment length polymorphism (AFLP) markers in a X. tropicalis interstrain hybrid. A total of 53 markers, including 51 AFLP markers and 2 isozyme markers, are presently assigned to 13 multipoint linkage groups on a map of the maternal strain, whereas 9 AFLP markers from the paternal strain are assigned to 3 linkage groups on a separate map. A dense genetic linkage map is essential in mapping new developmental mutants and determining their sequences by positional cloning.     

Autoantibody level modification in adult patients with idiopathic thrombocytopenic purpura following intravenous immunoglobulin treatment

The aim of this study was to determine whether treatment of patients with immune thrombocytopenic purpura (ITP) with intravenous immunoglobulin (IVIg) is associated with a modification in the antiplatelet glycoprotein (GP) antibodies (Abs). Fourteen patients with ITP (11 females and 3 males, mean age 36.6 years, range 18-72) received one to four IVIg treatment courses. The preparation used was ISIVEN that was given in a dose of 2 g/kg body weight in a 5-day schedule and in monthly intervals. Levels of IgG, IgM and IgA isotypes of Abs to GPs IIb/IIIa and Ib/IX were measured before the treatment, and before and after each treatment course. Two patients did not respond to IVIg, 6 had a temporary response, 5 had a sustained response and 1 patient responded well to the treatment but was lost to follow-up. The patients had a high prevalence of serum Abs directed against GPs IIb/IIIa and Ib/IX before the treatment, and the mean IgG isotype levels of both Abs increased after each treatment course, and decreased again before the following course began. Whenever high Ab levels of either isotype (> 10 U/ml) were detected before the treatment, they were significantly decreased before the last treatment course. The elevated levels of IgG Abs to IIb/IIIa and Ib/IX after every course are probably a result of displacement of these Abs from Fc receptors by the IVIg, rather than of exogenous infusion of these Abs contained within the IVIg, whereas the decrease in high Ab levels after a few treatment courses results from the immunomodulatory effects of IVIg: suppression of Ab formation, and the presence of anti-idiotypes.     

Analysis of bovine herpesvirus 4 (DN 599) major antigens with monoclonal antibodies and polyclonal immune serum

Summary Monoclonal antibodies (MAbs) and polyclonal immune sera were produced and used to identify the major antigens of bovine herpesvirus type 4 (BHV-4). SDS-polyacrylamide gel electrophoresis of immunoprecipitates of radiolabeled lysates from infected cells resolved 24 peptide bands varying from 12kDa to over 300kDa. Six peptides were identified as major viral antigens by immunoprecipitation. Based on the pattern of radioimmunoprecipitation, MAbs were assigned into four groups. Group 1 precipitated a tunicamycinsensitive glycoprotein complex which contained six components (245, 190, 152, 123, and 48/46kDa). Deglycosylation with endoglycosidase F revealed two peptides with M_r of 93 and 38kDa as the basic peptides of the glycoprotein complex. In addition, a 115kDa glycopeptide containing glycan-peptide bonds of mixed type was identified. Group 2 precipitated a non-glycosylated protein complex consisting of three monomers (33/31/30kDa). Groups 3 and 4 reacted with single monomeric non-glycosylated peptides with M_r of 48 and 14kDa, respectively. Although none of the MAbs exhibited significant neutralizing activity, some reacted strongly in immunosorbent and/or immunohistochemical assays, suggesting they may be good candidates for use in diagnostic assays.