Most nuclear systemic autoantigens are extremely disordered proteins: implications for the etiology of systemic autoimmunity

Patients with systemic autoimmune diseases usually produce high levels of antibodies to self-antigens (autoantigens). The repertoire of common autoantigens is remarkably limited, yet no readily understandable shared thread links these apparently diverse proteins. Using computer prediction algorithms, we have found that most nuclear systemic autoantigens are predicted to contain long regions of extreme structural disorder. Such disordered regions would generally make poor B cell epitopes and are predicted to be under-represented as potential T cell epitopes. Consideration of the potential role of protein disorder may give novel insights into the possible role of molecular mimicry in the pathogenesis of autoimmunity. The recognition of extreme autoantigen protein disorder has led us to an explicit model of epitope spreading that explains many of the paradoxical aspects of autoimmunity – in particular, the difficulty in identifying autoantigen-specific helper T cells that might collaborate with the B cells activated in systemic autoimmunity. The model also explains the experimentally observed breakdown of major histocompatibility complex (MHC) class specificity in peptides associated with the MHC II proteins of activated autoimmune B cells, and sheds light on the selection of particular T cell epitopes in autoimmunity. Finally, the model helps to rationalize the relative rarity of clinically significant autoimmunity despite the prevalence of low specificity/low avidity autoantibodies in normal individuals.     

Deficiencies in gut NK cell number and function precede diabetes onset in BB rats

Defects in the intestinal immune system may contribute to the pathogenesis of autoimmune diseases. Intraepithelial lymphocytes represent a substantial fraction of gut-associated lymphocytes, but their function in mucosal immunity is unclear. A newly described population of NK cells that spontaneously secrete IL-4 and IFN-gamma is present in the intraepithelial lymphocyte compartment of the rat. We hypothesized that defects in the number or function of these cells would be present in rats susceptible to autoimmunity. We report that the number of NKR-P1A(+)CD3(-) intraepithelial NK (IENK) cells is deficient before onset of spontaneous autoimmune diabetes in diabetes-prone BB (BBDP) rats. The absolute number of recoverable IENK cells was only approximately 8% of that observed in WF rats. Bone marrow transplantation from histocompatible WF donors reversed the IENK cell deficiency (and prevented diabetes) in these animals, suggesting a hemopoietic origin for their IENK cell defect. Analysis of diabetes-resistant BB rats, which develop autoimmune diabetes only after perturbation of the immune system, revealed IENK cell numbers intermediate between that of BBDP and WF rats. IENK cells were selectively depleted during treatment to induce diabetes. Prediabetic BBDP and diabetes-resistant BB animals also exhibited defective IENK cell function, including decreased NK cell cytotoxicity and reduced secretion of IL-4 and IFN-gamma. IENK functional defects were also observed in LEW and BN rats, which are susceptible to induced autoimmunity, but not in WF, DA, or F344 rats, which are resistant. Defects in IENK cell number and function may contribute to the pathogenesis of autoimmune diseases including type 1 diabetes.     

Gastrointestinal parasites of the guenons of western Uganda

From January 1998 to December 2002, we collected 293 fecal samples from free-ranging individuals of the 4 guenon species of western Uganda, i.e., redtail guenons (Cercopithecus ascanius), blue monkeys (Cercopithecus mitis), l'hoesti monkeys (Cercopithecus lhoesti), and vervet monkeys (Cercopithecus aethiops), to quantify the prevalence of gastrointestinal parasites. Helminth eggs, larvae, and protozoan cysts were isolated by sodium nitrate flotation and fecal sedimentation. Helminth parasites were identified, and infection prevalence was determined for all 4 guenon species. Coprocultures facilitated identification of strongylate nematodes. For the most common species, the redtail guenon, we documented prevalence of protozoan parasites and examined the effect of season and host sex on infection prevalence. Six nematodes (Strongyloidesfulleborni, Oesophagostomum sp., unidentified strongyle, Trichuris sp., Streptopharagus sp., and Enterobius sp.), 1 cestode (Bertiella sp.), 1 trematode (Dicrocoeliidae), and 5 protozoans (Entamoeba coli, Entamoeba histolytica, lodameoba butschlii, Giardia lamblia, and Chilomastix mesnili) were detected. Seasonal patterns of infection were not readily apparent for any parasite species infecting redtail guenons. Although prevalence never differed between male and female guenons, only adult females were infected with Oesophagostomum sp. and S. fulleborni.     

Hematozoa of the avian family Brachypteraciidae (the ground-rollers)

The Brachypteraciidae is an avian family endemic to Madagascar. Members of this family were mist-netted in Madagascar, and blood smears were made to screen for the presence of hematozoa. Smears were stained with Giemsa and examined at x100, x160, and x1000 for hematozoa. Three new species of avian hematozoa from wild-caught ground-rollers in Madagascar are described. Haemoproteus goodmani n. sp. is found in the pitta-like ground-roller (Atelornis pittoides), whereas Haemoproteus forresteri n. sp. and Leucocytozoon frascai n. sp. are from the rufous-headed ground-roller (Atelornis crossleyi). These represent the first hematozoa described from this family.     

Operational concept for the improved synthesis of (R)-3,3'-furoin and related hydrophobic compounds with benzaldehyde lyase

Biphasic reaction systems for enzyme catalysis are an elegant way to overcome limited solubility and stability of reactants and facilitate continuous processes. However, many synthetically useful enzymes are not stable in biphasic systems of water and organic solvent. The entrapment in polymer beads of polyvinyl alcohol has been shown to enable the stable operation of enzymes unstable in conventional biphasic reaction systems. We report the extension of this concept to continuous operation in a fluidised bed reactor. The enzyme benzaldehyde lyase was used for the continuous synthesis of enantiopure (R)-3,3'-furoin. The results show enhanced stability with half-life times under operation conditions of more than 100 h, as well as superior enzyme utilisation in terms of productivity. Furthermore, racemisation and oxidation of the product could be successfully prevented under the non-aqueous and inert reaction conditions.     

Construction, database integration, and application of an Oenothera EST library

Coevolution of cellular genetic compartments is a fundamental aspect in eukaryotic genome evolution that becomes apparent in serious developmental disturbances after interspecific organelle exchanges. The genus Oenothera represents a unique, at present the only available, resource to study the role of the compartmentalized plant genome in diversification of populations and speciation processes. An integrated approach involving cDNA cloning, EST sequencing, and bioinformatic data mining was chosen using Oenothera elata with the genetic constitution nuclear genome AA with plastome type I. The Gene Ontology system grouped 1621 unique gene products into 17 different functional categories. Application of arrays generated from a selected fraction of ESTs revealed significantly differing expression profiles among closely related Oenothera species possessing the potential to generate fertile and incompatible plastid/nuclear hybrids (hybrid bleaching). Furthermore, the EST library provides a valuable source of PCR-based polymorphic molecular markers that are instrumental for genotyping and molecular mapping approaches.     

Partial versus full allogeneic hemopoietic chimerization is a preferential means to inhibit type 1 diabetes as the latter induces generalized immunosuppression

In both humans and NOD mice, particular combinations of MHC genes provide the primary risk factor for development of the autoreactive T cell responses causing type 1 diabetes (T1D). Conversely, other MHC variants can confer dominant T1D resistance, and previous studies in NOD mice have shown their expression on hemopoietically derived APC is sufficient to induce disease protection. Although allogeneic hemopoietic chimerization can clearly provide a means for blocking T1D development, its clinical use for this purpose has been obviated by a requirement to precondition the host with what would be a lethal irradiation dose if bone marrow engraftment is not successful. There have been reports in which T1D-protective allogeneic hemopoietic chimerization was established in NOD mice that were preconditioned by protocols not including a lethal dose of irradiation. In most of these studies, virtually all the hemopoietic cells in the NOD recipients eventually converted to donor type. We now report that a concern about such full allogeneic chimeras is that they are severely immunocompromised potentially because their T cells are positively selected in the thymus by MHC molecules differing from those expressed by the APC available in the periphery to activate T cell effector functions. However, this undesirable side effect of generalized immunosuppression is obviated by a new protocol that establishes without a lethal preconditioning component, a stable state of mixed allogeneic hemopoietic chimerism sufficient to inhibit T1D development and also induce donor-specific tolerance in NOD recipients.     

TLR agonists abrogate costimulation blockade-induced prolongation of skin allografts

Costimulation blockade protocols are effective in prolonging allograft survival in animal models and are entering clinical trials, but how environmental perturbants affect graft survival remains largely unstudied. We used a costimulation blockade protocol consisting of a donor-specific transfusion and anti-CD154 mAb to address this question. We observed that lymphocytic choriomeningitis virus infection at the time of donor-specific transfusion and anti-CD154 mAb shortens allograft survival. Lymphocytic choriomeningitis virus 1) activates innate immunity, 2) induces allo-cross-reactive T cells, and 3) generates virus-specific responses, all of which may adversely affect allograft survival. To investigate the role of innate immunity, mice given costimulation blockade and skin allografts were coinjected with TLR2 (Pam3Cys), TLR3 (polyinosinic:polycytidylic acid), TLR4 (LPS), or TLR9 (CpG) agonists. Costimulation blockade prolonged skin allograft survival that was shortened after coinjection by TLR agonists. To investigate underlying mechanisms, we used "synchimeric" mice which circulate trace populations of anti-H2b transgenic alloreactive CD8+ T cells. In synchimeric mice treated with costimulation blockade, coadministration of all four TLR agonists prevented deletion of alloreactive CD8+ T cells and shortened skin allograft survival. These alloreactive CD8+ T cells 1) expressed the proliferation marker Ki-67, 2) up-regulated CD44, and 3) failed to undergo apoptosis. B6.TNFR2-/- and B6.IL-12R-/- mice treated with costimulation blockade plus LPS also exhibited short skin allograft survival whereas similarly treated B6.CD8alpha-/- and TLR4-/- mice exhibited prolonged allograft survival. We conclude that TLR signaling abrogates the effects of costimulation blockade by preventing alloreactive CD8+ T cell apoptosis through a mechanism not dependent on TNFR2 or IL-12R signaling.     

Kap120 functions as a nuclear import receptor for ribosome assembly factor Rpf1 in yeast

The nucleocytoplasmic exchange of macromolecules is mediated by receptors specialized in passage through the nuclear pore complex. The majority of these receptors belong to the importin beta protein family, which has 14 members in Saccharomyces cerevisiae. Nine importins carry various cargos from the cytoplasm into the nucleus, whereas four exportins mediate nuclear export. Kap120 is the only receptor whose transport cargo has not been found previously. Here, we characterize Kap120 as an importin for the ribosome maturation factor Rpf1, which was identified in a two-hybrid screen. Kap120 binds directly to Rpf1 in vitro and is released by Ran-GTP. At least three parallel import pathways exist for Rpf1, since nuclear import is defective in strains with the importins Kap120, Kap114, and Nmd5 deleted. Both kap120 and rpf1 mutants accumulate large ribosomal subunits in the nucleus. The nuclear accumulation of 60S ribosomal subunits in kap120 mutants is abolished upon RPF1 overexpression, indicating that Kap120 does not function in the actual ribosomal export step but rather in import of ribosome maturation factors.     

Independent evolution of functional MHC class II DRB genes in New World bat species

Genes of the major histocompatibility complex (MHC) play a pivotal role in the vertebrate immune system and are attractive markers for functional, fitness-related, genetic variation. Although bats (Chiroptera) represent the second largest mammalian order and are prone to various emerging infectious diseases, little is known about MHC evolution in bats. In the present study, we examined expressed MHC class II DRB sequences (exons 1 to 4) of New World bat species, Saccopteryx bilineata, Carollia perspicillata, Noctilio albiventris and Noctilio leporinus (only exon 2). We found a wide range of copy number variation of DRB loci with one locus detected in the genus Noctilio and up to ten functional loci observed in S. bilineata. Sequence variation between alleles of the same taxa was high with evidence for positive selection. We found statistical support for recombination or gene conversion events among sequences within the same but not between bat species. Phylogenetic relationships among DRB alleles provided strong evidence for independent evolution of the functional MHC class II DRB genes in the three investigated species, either by recent gene duplication, or homogenization of duplicated loci by frequent gene conversion events. Phylogenetic analysis of all available chiropteran DRB exon 2 sequences confirmed their monophyletic origin within families, but revealed a possible trans-species mode of evolution pattern in congeneric bat species, e.g. within the genera Noctilio and Myotis. This is the first study investigating phylogenetic relationships of MHC genes within bats and therefore contributes to a better understanding of MHC evolution in one of the most dominant mammalian order.