Phosphorylation of bid by casein kinases I and II regulates its cleavage by caspase 8.

Bid plays an essential role in Fas-mediated apoptosis of the so-called type II cells. In these cells, following cleavage by caspase 8, the C-terminal fragment of Bid translocates to mitochondria and triggers the release of apoptogenic factors, thereby inducing cell death. Here we report that Bid is phosphorylated by casein kinase I (CKI) and casein kinase II (CKII). Inhibition of CKI and CKII accelerated Fas-mediated apoptosis and Bid cleavage, whereas hyperactivity of the kinases delayed apoptosis. When phosphorylated, Bid was insensitive to caspase 8 cleavage in vitro. Moreover, a mutant of Bid that cannot be phosphorylated was found to be more toxic than wild-type Bid. Together, these data indicate that phosphorylation of Bid represents a new mechanism whereby cells control apoptosis.

A bacterial signal peptide directs efficient secretion of eukaryotic proteins in the baculovirus expression system.

Escherichia coli remains an organism of choice for the production of recombinant proteins required in large quantities. Whenever possible, secretion is the preferred strategy since it permits easy and efficient purification from the extracellular medium. Our efforts to use E. coli to secrete a human CD23 soluble variant fused to a pair of IgG binding domains via the Staphylococcal protein A signal peptide were unsuccessful. Surprisingly, when the same construct was expressed in the baculovirus system, efficient secretion was observed and cleavage of the signal peptide occurred at the expected site. Varying the genes in the fusions or the tags, or the topology of the gene and the tag, did not affect the high-level secretion and cleavage at the correct site. We envision that fusion of the bacterial signal sequence to eukaryotic recombinant genes will prove to be a tool of value for efficient protein secretion in insect cells using the baculovirus expression system.

Dissecting processing and apoptotic activity of a cysteine protease by mutant analysis.

We have compared the behavior of wild-type mouse NEDD-2, a neural precursor cell-expressed, developmentally down-regulated cysteine protease gene, to various mutant forms of the gene in both apoptotic activity in neuronal cells and proteolytic cleavage in the Semliki Forest virus and rabbit reticulocyte protein expression systems. Our results confirm that NEDD-2 processing and apoptotic activity are linked phenomena. They identify aspartate residues as likely targets for autocatalytic cleavage. They establish that cleavage events only occur at specific sites. Finally, they pinpoint differential effects of individual mutations on the overall proteolytic cleavage patterns, raising interesting questions related to the mechanisms of subunit assembly.

Interaction of heterozygous beta (0)-thalassemia and triplicated alpha globin loci in a Swiss-Spanish family.

We report a Swiss-Spanish family three members of which have the clinical picture of thalassemia intermedia. Restriction endonuclease mapping of the alpha-globin cluster and digestion with Mae I of the in vitro amplified 5' segment of the beta-globin gene shows a combination of triplicated alpha globin locus, anti-3.7 kb type, with heterozygous codon 39 C----T beta (0) thalassemic mutation. These, as well as 16 similar cases reported in the literature, permit the following conclusion: a single extra alpha-globin gene gives rise to a clinically significant degree of dyserythropoietic anemia only when it interacts with a severe beta(+) or beta(0) thalassemic mutation.

Prenatal diagnosis of thalassemia and hemoglobinopathies in Switzerland.

During a 10-month period, 10 couples originating from Africa (3), the tropics (1) and the thalassemia-belt region (6), living in Switzerland, requested prenatal diagnosis of hemoglobinopathies. Hb SS (twice), Hb Bart's (Hydrops fetalis) and beta-thalassemia major were diagnosed either by gene mapping or by direct detection of the mutations in DNA amplified by the PCR procedure. Whenever it was possible to obtain fetal blood or tissue, diagnosis was confirmed. In one Vietnamese man, concomitant existence of alpha-thal 1 with beta-thalassemia resulted in an unusually high Hb level because of balanced alpha and beta globin synthesis. The 10 couples examined originated from 7 different countries and presented at least 7 different Hb pathologies. This variety of pathologies represents the main difficulty for prenatal diagnosis of hemoglobinopathies in a non-endemic country. A diagnostic approach to overcome this problem is developed.

[Screening for alpha-thalassemia using DNA analysis]. / Dépistage de l'alpha-thalassémie par analyse du DNA.

alpha-thalassemia was sought by gene mapping in 258 subjects selected on the basis of origin (25%), microcytosis (7%), or origin and microcytosis combined (64%). Abnormal fragments (Xba I/probe alpha) were found in 58 cases (22.5%). Using other restriction enzymes it was possible to determine the genotype alpha-/aa in 39 patients and the genotype alpha-/alpha- in 13 patients; 2 patients also exhibited hemoglobin H (alpha-/--) disease. alpha triplication anti-3.7 kb was found in 2 subjects and zeta-thalassemia in 2 other samples. 57 out of 58 patients originated from the thalassemia belt or from Africa. alpha-thalassemia is the most frequent hemoglobinopathy (21% of patients at risk) and hematologically is characterized by microcytosis. The Hb A2 level is decreased only in the alpha-/-- form of the disease. The main advantage of diagnosing zeta-thalassemias and alpha triplications lies in the possible clinical implications in the event of association with other hemoglobinopathies or beta-thalassemia.

Trypanosoma brucei gambiense: cerebral immunopathology in mice.

Ninety outbred white adult female mice were infected with Trypanosoma brucei gambiense (GUMS 2, alias LUMP 1237) originating from a Zairian patient and known to produce a low parasitaemia in rodents. The development of cerebral trypanosomiasis was independent upon the number of parasites inoculated per mouse. Trypanosomes appeared in the circulating blood about four months after infection, when some mice started to show the first signs of paresis which subsequently led to cachexia. A clinical test to stage such a development is described. 57 mice were sacrificed at various intervals after infection, starting from one to 22 months. The morphological changes in the brain consisted of a diffuse meningoencephalitis in 45 mice, (78.9%) often associated with parasites, the latter being best visualised in 21 mice (36.8%) by immunofluorescence using a specific antitrypanosome antibody. The trypanosomes showed a predominantly extravascular distribution in the cerebral parenchyma, to a lesser extent in the meninges and only rarely in the choroid plexuses. Deposits of immunoglobulins in the choroid plexuses and cerebral infiltrations by plasma cells were mild. The level of circulating immune complexes was found to be increased. Adequate intravenous Melarsoprol did not prevent the disease from progressing to advanced stages, and there is limited morphological evidence that it did not eradicate the parasite from the host. The immunofluorescent use of an antitrypanosome antibody to demonstrate the persistence of tissue parasites after chemotherapy is recommended. Murine models seem therefore to be suitable for drug screening in cerebral trypanosomiasis since all three trypanosomes of the brucei group can be adapted to mice.

Deposition of idiotype-anti-idiotype immune complexes in renal glomeruli after polyclonal B cell activation.

We investigated the possible role of idiotypic interactions in the pathogenesis of the glomerular lesions observed in mice undergoing polyclonal B cell activation. BALB/c mice were studied for the presence of renal deposits of T15 idiotype-anti-T15 idiotype-immune complexes (IC) after injection of bacterial lipopolysaccharides (LPS). The T15 idiotype is the major idiotype of BALB/c mice anti-phosphorylcholine (PC) antibodies, which are cross-reactive with the idiotype of the TEPC-15 myeloma protein. This model was used because T15 idiotype-anti-T15 idiotype IC have been detected in the circulation of BALB/c mice after polyclonal B cell activation. First, an idiotype-specific immunofluorescence technique allowed us to detect T15 idiotype-bearing immunoglobulins in glomeruli from day 6 to day 28 after LPS injection. Second, fluorescein isothiocyanate-conjugated TEPC-15 myeloma protein was found to localize in the glomeruli after in vivo injection 18 d after LPS administration. This renal localization was shown to be idiotype-specific and could be quantified in a trace-labeling experiment. Third, kidney-deposited immunoglobulins of mice injected with LPS were eluted, radiolabeled, and analyzed by radioimmunoassay. Both T15 idiotype-bearing immunoglobulins and anti-T15 idiotype antibodies were detected in the eluates, providing further evidence for a renal deposition of T15 idiotype-anti-T15 idiotype IC. Polyclonal B cell activation is likely to result in a simultaneous triggering of many idiotypic clones and of corresponding anti-idiotypic clones represented in the B cell repertoire. This could lead to the formation of a variety of idiotype-anti-idiotype IC that could participate in the development of glomerular lesions.

Trypanosoma brucei brucei: the response to Melarsoprol in mice with cerebral trypanosomiasis. An immunopathological study.

A murine model for cerebral trypanosomiasis was adapted to study the efficacy of Melarsoprol which was apparently curative in high intravenous doses (10 mg/kg, 3 x 10 mg/kg, 20 mg/kg) in advanced infection (6th week); only one relapse occurred, but observation time was limited. Deposits of Ig and C3 in the choroid plexuses tended to disappear after successful treatment. Circulating immune complexes increased in the 1st week after therapy and returned to normal values in the 2nd week. Such an increase could temporarily be prevented by chloroquine, which may explain the reported reduction of side-effects from Melarsoprol in man after use of chloroquine in sleeping sickness. Melarsoprol (3 x 3.6 mg/kg) given intraperitoneally showed apparent cures but also relapses. Melarsoprol in high intraperitoneal doses (3 x 10 mg/kg) showed an increasing number of relapses if related to the duration of infection. Morphologically, a diffuse interstitial distribution of the parasites appeared in the CNS after relapse had occurred (shift), contrasting with the preferential localization of the parasites in the choroid plexuses of untreated mice. Such a shift was best visualized by immunofluorescence using specific antitrypanosomal antibodies. Relapse-mice invariably showed increased levels of circulating immune complexes suggesting this serological test for early detection of relapses. Tissue parasites appeared to be a likely cause of relapses, irrespective of the duration of infection. A short observation time with no circulating blood parasites is no guarantee of cure. Benznidazole (3 x 1 g/kg) was ineffective in advanced infection.

Immunopathological aspects of Plasmodium berghei infection in five strains of mice. II. Immunopathology of cerebral and other tissue lesions during the infection.

Histological changes during the course of P. berghei infection were investigated in A/J, BALB/c, OF1, CBA and C57B1 mice. The findings were studied in relation to serological aspects (Contreras et al., 1980). High mortality and acute deaths occurred in A/J, BALB/c and OF1 mice and marked cerebral lesions were found in these strains from day 15, including congestion of meningeal and cerebral veins and capillaries, blocking of these vessels by heavily parasitized RBC, cerebral oedema and haemorrhages. Such lesions were minimal in CBA and C57B1 mice, and absent in mice examined 21 and 24 days after infection. Small deposits of IgG and traces of C3 were detected by immunofluorescence in the choroid plexus of most mice from day 9. Renal lesions included congestion, plugging of veins and capillaries, low-grade mononuclear infiltration and mesangial thickening; these changes were most marked in CBA, C57B1 and A/J mice. Glomerular deposits of IgM were present in all strains in the first week of infection. IgG and C3 were detected in the second week, but only traces were found in CBA mice. The livers showed congestion, accumulation of pigment in swollen Kupffer cells and mononuclear portal infiltration; these were most pronounced in A/J mice. In the spleen, there was a great increase in the reticuloendothelial cell population, white pulp proliferation, congestion and accumulation of pigment and plasma cell reaction; the pattern of white pulp expansion varied in the different strains. The results suggest that cerebral lesions play a significant role in the aetiology of acute deaths in this malaria model.

Trypanosoma brucei brucei: a model for cerebral trypanosomiasis in mice--an immunological, histological and electronmicroscopic study.

The successful induction of cerebral trypanosomiasis in ordinary laboratory mice using Trypanosoma brucei brucei is reported. Sequential studies demonstrated the presence of trypanosomes in the interstitium of the choroid plexus at the fourth week after infection which correlated with the appearance of anti-trypanosomal antibodies, a rise of IgM and IgG serum levels and a rise of Clq binding activity as well as a decrease of C3 levels. Electronmicroscopic studies showed that the parasites were flagellated and localized extracellularly mainly in the interstitium of the choroid plexus. Granular immunofluorescent deposits of Ig and C3 were most marked in the choroid plexus. Electron-dense deposits suggestive of immune complexes were seen in subendothelial, interstitial and subependymal areas of the choroid plexus. Since autoantibodies to the brain were found in the serum of some mice, the possible involvement of autoimmune manifestations in the pathogenesis of cerebral lesions has to be considered. The pattern of inflammatory foci at the eighth week after infection was very similar to that observed in cerebral African trypanosomiasis in man. After treatment with ethidium bromide, trypanosomes persisted in the tissues when circulating parasites could no longer be detected. These observations suggest a sequential involvement of brain structures during African trypanosomiasis. Trypanosomes may first migrate from the vascular compartment into the interstitium of the choroid plexus, possible favoured by increased vascular permeability. Circulating immune complexes and complement activation may be involved at this state. Trypanosomes localized in the choroid plexus may then trigger a local immunologically mediated inflammatory reaction favouring the migration of trypanosomes into the CSF and further invasion of other cerebral structures.

A model for cardiopathy induced by Trypanosoma brucei brucei in mice. A histologic and immunopathologic study.

The successful induction of pancarditis in mice by the use of Trypanosoma brucei brucei is reported. The sequential analysis of whole-organ sections demonstrated the presence of trypanosomes in the cardiac structures from the fourth week after infection. Parasites predominated on the endocardial and epicardial side but were also present in the valves, the conducting system, and the lymphatic system draining the heart, the latter being particularly evident in late infection. At the time of parasite invasion, deposits of IgM and IgG and of complement (C3) appeared in the tissues. Also at this time parasitemia reached a plateau, and the circulating specific antitrypanosomal antibodies, the serum Ig and C3, as well as the Clq activity, reached pathologic levels. Cellular response followed parasite invasion and appeared to be similar to that described in human African trypanosomiasis. In late infection, the draining lymph nodes showed a marked histiocytic proliferation, and the vessels became convoluted and distended. The suggested pathogenic mechanisms involve immunologic and mechanical factors. It is possible that the immunologic process prepares for a simultaneous or subsequent parasite invasion of the tissues with an associated inflammatory response. The partial obstruction of the lymphatic cardiac draining system probably accounts at least in part for the peculiar distribution of the parasite-induced lesions. A therapeutic trial was unsuccessful, but the persistence of trypanosomes in the tissues when circulating parasites were no longer detectable may account for relapses.

The role of the host immune response in the development of tissue lesions associated with African trypanosomiasis in mice.

A variety of tissue lesions occurs in African trypanosomiasis, in the pathogenesis of which direct toxic effects of the parasite as well as immunological mechanisms may be involved. The purpose of the present study was to evaluate the role of the host immune response in inducing tissue damage in this disease and particularly in the production of lesions in striated muscle. The development of muscle lesions in T. brucei infection was studied in several groups of mice with different forms of immunodeficiency, as well as in normal mice. In the normal mice, foci of intense inflammation and necrosis were found in the cardiac and skeletal muscles 2 weeks or more after infection. In these lesions, there was a heavy deposition of IgG and IgM, and of trypanosomal antigens. In irradiated, newborn mice, and athymic nude mice infected with T. brucei, these inflammatory lesions were not found, although large numbers of trypanosomes were present between the muscle fibres. The characteristic lesions could be induced in athymic nude mice by transfer of normal spleen cells or of normal T lymphocytes 1 week after the onset of infection. The lesions were also partly induced by transfer of antibody to T. brucei. No antibodies to tissue components, particularly to cardiac myofibrils, were found in any of the infected mice. The results of this study show that immunodeficiency suppresses the development of the characteristic muscle lesions of African trypanosomiasis. The relative importance of humoral and cellular immune mechanisms in the pathogenesis of these lesions is not year clear.