Effect of osteopontin alleles on beta-glucan-induced granuloma formation in the mouse liver

The granuloma formation is a host defense response against persistent irritants. Osteopontin is centrally involved in the formation of granulomas. Three osteopontin alleles, designated a, b, and c, have been found in mice. Here we used a murine model of zymosan (beta-glucan)-induced granuloma formation in the liver to determine possible functional differences between the osteopontin alleles in cell-mediated immunity. In contrast to mice with alleles a or c, mice with the allele b was defective in granuloma formation. As detected by mRNA expression, cytokines and chemokines known to be critically involved in granuloma formation were elicited in liver tissue, regardless of the osteopontin allele expressed. Alignment of the deduced amino acid sequences showed that unlike osteopontin c, b differs from a in 11 amino acids. All three osteopontin alleles had normal cell-binding properties. However, only the b allelic form was defective in the induction of cell migration as tested with dendritic cells. In conclusion, generation of a granulomatous response in mice depends critically on the presence of a functional osteopontin allele. Defective granuloma formation in mice with allele b is likely to be because of an impaired chemotactic function of the osteopontin b protein on immunocompetent cells.     

Antigen-specific T cell suppression by human CD4+CD25+ regulatory T cells

Anergic/suppressive CD4+CD25+ T cells have been proposed to play an important role in the maintenance of peripheral tolerance. Here we demonstrate that in humans these cells suppress proliferation to self antigens, but also to dietary and foreign antigens. The suppressive CD4+CD25+ T cells display a broad usage of the T cell receptor Vbeta repertoire,suggesting that they recognize a wide variety of antigens. They reside in the primed/memory CD4+CD45RO+CD45RB(low) subset and have short telomeres, indicating that these cells have the phenotype of highly differentiated CD4+ T cells that have experienced repeated episodes of antigen-specific stimulation in vivo. This suggests that anergic/suppressive CD4+CD25+ T cells may be generated in the periphery as a consequence of repeated antigenic encounter. This is supported by the observation that highly differentiated CD4+T cells can be induced to become anergic/suppressive when stimulated by antigen presented by non-professional antigen-presenting cells. We suggest that besides being generated in the thymus, CD4+CD25+ regulatory T cells may also be generated in the periphery. This would provide a mechanism for the generation of regulatory cells that induce tolerance to a wide array of antigens that may not be encountered in the thymus.     

Juvenile rhesus monkeys have more colonic granulomas than adults after primary infection with<Emphasis Type="Italic"> Schistosoma mansoni</Emphasis>

Adults and children have differences in their susceptibility to schistosomiasis. Whether this age-dependent innate susceptibility influences parasite-caused granulomogenesis is difficult to assess in humans. Therefore, we exposed juvenile and adult female rhesus monkeys to primary infection with Schistosoma mansoni. Hepatic and intestinal granuloma formation was observed in both pre-pubescent and adult monkeys. Two distinct stages of granulomas were discerned, the exudative and the productive stage. In the intestine, more granulomas were generated in the colon than in the ileum. In contrast to the adult animals, the juvenile rhesus monkeys had higher numbers of colonic granulomas, these higher numbers being predominantly of the more advanced productive stage. Juvenile animals had a statistically non-significant increased worm burden. These results suggest that juvenile rhesus monkeys have a significantly more intense and advanced colonic response towards entrapped S. mansoni eggs after primary schistosome infections and, thereby, are more susceptible to parasite infection.Research protocols involving non-human primates received ethical clearance by the Institutional Animal Care and Use Committee of the Biomedical Primate Research Centre (Rijswijk, The Netherlands), according to Dutch Law.     

Aβ40 is a major form of β-amyloid in nonhuman primates

Because aged nonhuman primates show β-amyloid (Aβ) deposition in senile plaques and blood vessels similar to that seen in human aging and AD, we used C-terminal specific antibodies to Aβ₄₀ and Aβ₄₂ to investigate Aβ peptide length in the brains of 11 aged rhesus monkeys and a 59-year-old chimpanzee. In contrast to AD, where the earliest and most prominent form of Aβ in senile plaques is Aβ₄₂, in the monkey, Aβ₄₀-positive plaques predominated. The ratio of Aβ₄₎: Aβ₄₂-positive plaques averaged 2.08 in the monkey, as compared to a mean ratio of 0.37 in 68 human AD subjects (p < 0.001). Aβ₄₀ was also more prominent in the chimpanzee than in humans. Possible explanations for these findings include species differences in the cleavage of Aβ from the amyloid precursor protein or in the activity of a putative carboxy peptidase forming Aβ₄₀ from Aβ₄₂ in situ.     

Fusion with mouse thymocytes leads to expression of p30 antigen in fibroblasts of BALB/Mo mice

Embryonic fibroblasts of BALB/Mo mice carrying the endogenous genome of Moloney murine leukemia virus (M-MuLV) in addition to murine type C viruses were fused with Swiss mouse thymocytes, B lymphocytes, macrophages, or embryonic fibroblasts. We wanted to determine whether these cells contained physiological factors involved in induction of the integrated viral genome(s). Fusion with thymocytes and, to a lesser extent, fusion with B lymphocytes induced viral genome expression as demonstrated by the appearance of viral structural protein p30 detected by immunofluorescence. Maximal expression of p30 was observed about 36 hr after the fusion event, using thymocytes from 1- to 2-week-old Swiss mice. This temporary expression of p30 was not accompanied by release of infectious virus. Fusion of BALB/Mo fibroblasts with Swiss mouse macrophages or embryonic fibroblasts led to neither the production of p30 nor the release of detectable infectious virus. These results suggest the existence of thymocyte-specific and possibly B lymphocyte-specific factor(s) involved in the control of expression of integrated viral genome(s).     

Accumulation of very long-chain fatty acids does not affect mitochondrial function in adrenoleukodystrophy protein deficiency

X-linked adrenoleukodystrophy (X-ALD, OMIM 300100) is a severe inherited neurodegenerative disease, associated with the accumulation of very long-chain fatty acids (VLCFA). The recent unexpected observation that the accumulation of VLCFA in tissues of the Abcd1-deficient mouse model for X-ALD is not due to a deficiency in VLCFA degradation, led to the hypothesis that mitochondrial abnormalities might contribute to X-ALD pathology. Here, we report that in spite of substantial accumulation of VLCFA in whole muscle homogenates, normal VLCFA levels were detected in mitochondria obtained by organellar fractionation. Polarographic analyses of the respiratory chain as well as enzymatic assays of isolated muscle mitochondria revealed no differences between X-ALD and control mice. Moreover, analysis by electron microscopy, revealed normal size, structure and localization of mitochondria in muscle of both groups. Similar to the results obtained in skeletal muscle, the mitochondrial enzyme activities in brain homogenates of Abcd1-deficient and wild-type animals also did not differ. Finally, studies on mitochondrial oxidative phosphorylation in permeabilized human skin fibroblasts of X-ALD patients and controls revealed no abnormalities. Thus, we conclude that the accumulation of VLCFA per se does not cause mitochondrial abnormalities and vice versa-mitochondrial abnormalities are not responsible for the accumulation of VLCFA in X-ALD mice.     

Autoimmunity and inflammation due to a gain-of-function mutation in phospholipase C gamma 2 that specifically increases external Ca2+ entry

The identification of specific genetic loci that contribute to inflammatory and autoimmune diseases has proved difficult due to the contribution of multiple interacting genes, the inherent genetic heterogeneity present in human populations, and a lack of new mouse mutants. By using N-ethyl-N-nitrosourea (ENU) mutagenesis to discover new immune regulators, we identified a point mutation in the murine phospholipase Cg2 (Plcg2) gene that leads to severe spontaneous inflammation and autoimmunity. The disease is composed of an autoimmune component mediated by autoantibody immune complexes and B and T cell independent inflammation. The underlying mechanism is a gain-of-function mutation in Plcg2, which leads to hyperreactive external calcium entry in B cells and expansion of innate inflammatory cells. This mutant identifies Plcg2 as a key regulator in an autoimmune and inflammatory disease mediated by B cells and non-B, non-T haematopoietic cells and emphasizes that by distinct genetic modulation, a single point mutation can lead to a complex immunological phenotype.     

Mechanisms of cold sensitivity of paramyotonia congenita mutation R1448H and overlap syndrome mutation M1360V

Missense mutations of the human skeletal muscle voltage-gated Na⁺ channel (hSkM1) cause a variety of neuromuscular disorders. The mutation R1448H results in paramyotonia congenita and causes cold-induced myotonia with subsequent paralysis. The mutation M1360V causes an overlapping syndrome with both K⁺-induced muscle weakness and cold-induced myotonia. The molecular mechanisms of the temperature dependence of these disorders are not well understood. Therefore we investigated physiological parameters of these Na⁺ channel mutations at different temperatures. Channel proteins were recombinantly expressed in human embryonic kidney cells and studied electrophysiologically, using the whole-cell patch-clamp technique. We compared the wild-type (WT) channel with both mutants at different temperatures. Both mutations had slower inactivation and faster recovery from inactivation compared to WT channels. This effect was more pronounced at the R1448H mutation, leading to a larger depolarization of the cell membrane causing myotonia and paralysis. The voltage dependence of activation of R1448H was shifted to more negative membrane potentials at lower temperature but not at the M1360V mutation or in the WT. The window current by mutation R1448H was increased at lower temperatures. The results of this study may explain the stronger cold-induced clinical symptoms resulting from the R1448H mutation in contrast to the M1360V mutation.     

Electronic data processing in the Danish Cytogenetic Central Register and EDP problems of registers in general

A brief introduction to the Danish Cytogenetic Central Register (DCCR) is given, and possibilities, principles and problems concerning the establishment and maintenance of a national cytogenetic register are presented.
Various data carrier media for registers in general are discussed, of which the magnetic disc is considered most appropriate. General principles for programs capable of performing insertions, deletions and other modifications in the data base are outlined as well as the principles for the programs in the DCCR.
The individual records should preferably be identified by aid of a central person registration number (CPR) rather than by name. The data should be stored and sorted by this identification in order to facilitate retrieval of a desired record. The structure of the records is discussed with regard to prevention of the occurrence of certain errors as well as the optimization of processing.
Flexibility and economy of space are achieved by using programs able to handle records of unequal length, and problems occurring in connection with this are discussed. The question of how to protect sensitive data is dealt with, and two different methods used in the DCCR are outlined. Programs capable of analyzing karyotypes with the purpose of recognizing various cytogenetic syndromes have been developed for use in the DCCR. Various examples of computing times of typical program runs are presented.