Regulated activity of the IgH intron enhancer (E{micro}) in the T lymphocyte lineage

The activity of the IgH (E_µ) enhancer in the T lymphocytelineage has been investigated using both transgenic mice andtransfection studies. Thymocyte fractionation experiments indicatethat a transgene consisting of the bacterial chloramphenicolacetyl transferase (CAT) gene, linked to Eµ and the SV40early promoter (E_µ–CAT), is expressed only in thymocyteswith a mature medullary phenotype and not in immature cells.Transfection of this same construct into two thymoma cell linesrepresenting different stages of thymocyte development mimicsthe pattern of activity observed in vivo. Further transfectionexperiments suggest that this pattern of expression might beattributed to the differential activity of the E2E3 and octanucleotidemotifs of E_µ during development. In contrast, an Ig transgene(linked to E_µ and an Ig V promoter) is expressed in themajority of thymocytes. We envisage that the different patternsof expression of the two transgenes reflect interactions betweentheir respective promoters and the factors which are bound toE_µ at different stages of thymocyte development. Althoughdiffering in their pattern of expression within the thymus,the two transgenes share the property of extinction in peripheralT lymphocytes. These results indicate that the expression ofE_µ-linked transgenes in the thymus cannot simply be explainedby activation of the enhancer in a lymphoid progenitor cellprior to B/T lineage divergence. Rather, the enhancer (or componentsof it) must be independently activated (and inactivated) duringT lymphocyte development. Furthermore, this activity is consistentwith the developmental timing of Ig D_H–J_H rearrangementsin these cells.     

The Enterprise,a massive transposon carrying Spok meiotic drive genes

The genomes of eukaryotes are full of parasitic sequences known as transposable elements (TEs). Here, we report the discovery of a putative giant tyrosine-recombinase-mobilized DNA transposon, Enterprise, from the model fungus Podospora anserina. Previously, we described a large genomic feature called the Spok block which is notable due to the presence of meiotic drive genes of the Spok gene family. The Spok block ranges from 110 kb to 247 kb and can be present in at least four different genomic locations within P. anserina, despite what is an otherwise highly conserved genome structure. We propose that the reason for its varying positions is that the Spok block is not only capable of meiotic drive but is also capable of transposition. More precisely, the Spok block represents a unique case where the Enterprise has captured the Spoks, thereby parasitizing a resident genomic parasite to become a genomic hyperparasite. Furthermore, we demonstrate that Enterprise (without the Spoks) is found in other fungal lineages, where it can be as large as 70 kb. Lastly, we provide experimental evidence that the Spok block is deleterious, with detrimental effects on spore production in strains which carry it. This union of meiotic drivers and a transposon has created a selfish element of impressive size in Podospora, challenging our perception of how TEs influence genome evolution and broadening the horizons in terms of what the upper limit of transposition may be.

Transposable elements (TEs) are major agents of change in eukaryotic genomes. Their ability to selfishly parasitize their host replication machinery has large impacts on both genome size and on gene regulation (Chénais et al. 2012). In extreme cases, TEs can contribute up to 85% of genomic content (Schnable et al. 2009), and expansion and reduction of TEs can result in rapid changes in both genome size and architecture (Haas et al. 2009; Möller and Stukenbrock 2017; Talla et al. 2017). Generally, TEs have small sizes (∼50–12,000 bp) and accomplish these large-scale changes through their sheer number. For example, there are ∼1.1 million Alu elements in the human genome, which have had a large impact on genome evolution (Jurka 2004; Bennett et al. 2008). The largest known cases among Class I retrotransposons are long terminal repeat (LTR) elements that can be as large as 30 kb, but among Class II DNA transposons, Mavericks/Polintons are known to grow as large as 40 kb through the capture of additional open reading frames (ORFs) (Arkhipova and Yushenova 2019). Recently, a behemoth TE named Teratorn was described in teleost fish; it can be up to 182 kb in length, dwarfing all other known TEs. Teratorn has achieved this impressive size by fusing a piggyBac DNA transposon with a herpesvirus, thereby blurring the line between TEs and viruses (Inoue et al. 2017, 2018). Truly massive transposons may be lurking in the depths of many eukaryotic genomes, but the limitations of short-read genome sequencing technologies and the lack of population-level high-quality assemblies may make them difficult to identify.The Spok block is a large genomic feature that was first identified thanks to the presence of the spore killing (Spok) genes in species from the genus Podospora (Grognet et al. 2014; Vogan et al. 2019). The Spoks are selfish genetic elements that bias their transmission to the next generation in a process known as meiotic drive. Here, drive occurs by inducing the death of spores that do not inherit them, through a single protein that operates as both a toxin and an antidote (Grognet et al. 2014; Vogan et al. 2019). The first Spok gene described, Spok1, was discovered in Podospora comata (Grognet et al. 2014). In P. anserina, the homologous gene Spok2 is found at high population frequencies, whereas two other genes of the Spok family, Spok3 and Spok4, are at low to intermediate frequencies (Vogan et al. 2019). Unlike Spok1 and Spok2, however, Spok3 and Spok4 are always associated with a large genomic region (the Spok block). The Spok block can be located at different chromosomal locations in different individuals but is never found more than once in natural strains. The number of Spok genes and the location of the Spok block (which carries Spok3, Spok4, or both) define the overall meiotic driver behavior of a given genome, which can be classified into the so-called Podospora spore killers or Psks (van der Gaag et al. 2000; Vogan et al. 2019). The Spok block stands out not only because of its size, typically around 150 kb, but also because there is otherwise high genome collinearity among strains of P. anserina and with the related species P. comata and P. pauciseta (Vogan et al. 2019).The fact that the Spok block is found at unique genomic positions between otherwise highly similar strains is of prime interest as each novel Spok block position creates a unique meiotic drive type (Psk) due to the intricacies of meiosis in Podospora (Vogan et al. 2019). We therefore set out to determine the mechanism through which the Spok block relocates throughout the genome. Additionally, we annotated the gene content of the various Spok blocks to describe their composition and understand what represents the minimal component of the Spok block. Lastly, we conducted fitness assays to investigate whether the presence of the Spok block imparts any detrimental effects upon the host.     

Autoantibodies to C-reactive protein is a common finding in SLE,but not in primary Sjögren's syndrome,rheumatoid arthritis or inflammatory bowel disease

The occurrence of antibodies to human C-reactive protein (CRP) was analysed by enzyme-linked immunosorbent assay (ELISA) in 56 patient sera known to contain antibodies to double-stranded DNA (dsDNA) and in 16 sera from patients with primary Sj?gren's syndrome (SS), 15 rheumatoid arthritis, 31 Crohn's disease, and 37 ulcerative colitis. Eighty-seven per cent of the patients with anti-dsDNA antibodies had systemic lupus erythematosus (SLE) and the remaining had autoimmune hepatitis. The cut-off for positive anti-CRP test was set at the 95th percentile of 100 healthy blood donors. Twenty of 56 anti-dsDNA sera (36%) and two of 16 SS sera (13%) had antibodies reactive with human CRP, whereas all other samples were negative. Thirteen of 27 SLE patients (48%) were positive on at least one occasion. The sera containing anti-CRP antibodies only reacted with surface-bound antigen, but not with native CRP in solution. In conclusion, we found that autoantibodies to CRP are common in sera from patients with anti-dsDNA antibodies. It is not likely that this explains the relative failure of CRP response in patients with active SLE. However, it cannot be excluded that anti-CRP autoantibodies have other biological potentials of pathophysiological interest in SLE, for instance by binding to CRP deposited on cell and tissue surfaces.     

Whole-body imaging of sequestration of Plasmodium falciparum in the rat

The occlusion of vessels by packed Plasmodium falciparum-infected (iRBC) and uninfected erythrocytes is a characteristic postmortem finding in the microvasculature of patients with severe malaria. Here we have employed immunocompetent Sprague-Dawley rats to establish sequestration in vivo. Human iRBC cultivated in vitro and purified in a single step over a magnet were labeled with 99mtechnetium, injected into the tail vein of the rat, and monitored dynamically for adhesion in the microvasculature using whole-body imaging or imaging of the lungs subsequent to surgical removal. iRBC of different lines and clones sequester avidly in vivo while uninfected erythrocytes did not. Histological examination revealed that a multiadhesive parasite adhered in the larger microvasculature, inducing extensive intravascular changes while CD36- and chondroitin sulfate A-specific parasites predominantly sequester in capillaries, inducing no or minor pathology. Removal of the adhesive ligand Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), preincubation of the iRBC with sera to PfEMP1 or preincubation with soluble PfEMP1-receptors prior to injection significantly reduced the sequestration. The specificity of iRBC binding to the heterologous murine receptors was confirmed in vitro, using primary rat lung endothelial cells and rat lung cryosections. In offering flow dynamics, nonmanipulated endothelial cells, and an intact immune system, we believe this syngeneic animal model to be an important complement to existing in vitro systems for the screening of vaccines and adjunct therapies aiming at the prevention and treatment of severe malaria.     

Effect of surface treatment on the shear bond strength of three resin cements to a machinable feldspatic ceramic

The aim of this study was to evaluate the shear bond strength of three resin cements to Vita Mark II ceramics under different pretreatments of the ceramic surface and to examine whether simplified pretreatment procedures would result in satisfying results compared to the state of the art. RelyX Unicem (RXU), Calibra (CAL), and Variolink II (VAR) were used as resin cements and bonded to machine milled feldspatic disks, pretreated in five different ways. (1) no pretreatment of the ceramic surface; (2) surface etched with hydrofluoric acid (HF); (3) ceramic surface silanized; (4) ceramic surface etched (HF) and silanized, (5) ceramic surface etched (HF), silanized, and covered with Heliobond. The shear bond strengths were measured initially, after 5000 and 10,000 thermocycles (TC). After 10,000 TC for CAL only procedure 5 resulted in a reliable adhesion median value of 10.7 MPa. VAR showed median values of 24.6, 17.2, and 18.1 MPa for pretreatments 5, 3, and 4, respectively. RXU performed 25.9, 22.0, and 11.0 MPa for procedures 5, 4, and 3, respectively. For procedure 2, RXU revealed the significantly highest value with 15.4 MPa (U-test, p = 0.05). Only RXU-luted specimens of procedure 1 survived the 10,000 thermocycles. The results revealed that a simplification of the ceramic pretreatment for VAR and RXU might be possible.     

The life cycle of<Emphasis Type="Italic"> Anisakis simplex</Emphasis> in the Norwegian Deep (northern North Sea)

Copepoda (Calanus finmarchicus n=1,722, Paraeuchaeta norvegica n=1,955), Hyperiidae (n=3,019), Euphausiacea (Meganyctiphanes norvegica n=4,780), and the fishes Maurolicus muelleri (n=500) and Pollachius virens (n=33) were collected in the Norwegian Deep (northern North Sea) during summer 2001 to examine the importance of pelagic invertebrates and vertebrates as hosts of Anisakis simplex and their roles in the transfer of this nematode to its final hosts (Cetaceans). Third stage larvae (L3) of A. simplex were found in P. norvegica, M. muelleri and P. virens. The prevalence of A. simplex in dissected P. norvegica was 0.26%, with an intensity of 1. Prevalences in M. muelleri and P. virens were 49.6% and 100.0%, with mean intensities of 1.1–2.6 (total fish length 6.0–7.2) and 193.6, respectively. All specimens of C. finmarchicus and M. norvegica examined were free of anisakid nematode species and no other parasites were detected. P. norvegica, which harboured the third stage larvae, is the obligatory first intermediate host of A. simplex in the investigated area. Though there was no apparent development of larvae in M. muelleri, this fish can be considered as the obligatory second intermediate host of A. simplex in the Norwegian Deep. However, it is unlikely that the larva from P. norvegica can be successfully transmitted into the cetacean or pinniped final hosts, where they reach the adult stage. An additional growth phase and a second intermediate host is the next phase in the life cycle. Larger predators such as P. virens serve as paratenic hosts, accumulating the already infective stage from M. muelleri. The oceanic life cycle of A. simplex in the Norwegian Deep is very different in terms of hosts and proposed life cycle patterns of A. simplex from other regions, involving only a few intermediate hosts. In contrast to earlier suggestions, euphausiids have no importance at all for the successful transmission of A. simplex in the Norwegian Deep. This demonstrates that this nematode is able to select definite host species depending on the locality, apparently having a very low level of host specificity. This could explain the wide range of different hosts that have been recorded for this species, and can be seen as the reason for the success of this parasite in reaching its marine mammal final hosts in an oceanic environment.     

Dendritic cells transfected with cytopathic self-replicating RNA induce crosspriming of CD8+ T cells and antiviral immunity

A potential shortcoming of nonlive vaccines is their relative inefficiency in generating T cell responses, thus limiting their application in infections requiring cellular immunity. Here, we present a system to induce cellular immunity and to study the immunological implications of time-delayed dendritic cell (DC) apoptosis and antigen reprocessing in vivo. We generated a self-replicating cytopathic pestivirus RNA to enhance production and presentation of hepatitis C virus (HCV) antigens and to induce apoptosis in DC 24-48 hr after transfection. Replicon-transfected H-2(b) DCs used to immunize HLA-A2 transgenic mice induced protection upon challenge with a vaccinia virus expressing HCV antigens. Induction of cell death enhanced the immunogenicity of DC-associated antigen. Transfer of cellular material from vaccine DCs to endogenous antigen presenting cells was visualized in lymph nodes and spleen, and crossprimed CD8(+) T cells were characterized. The findings are relevant for the rational design of vaccines against noncytopathic pathogens like HCV.     

Single nucleotide variation analysis in 65 candidate genes for CNS disorders in a representative sample of the European population

The detailed investigation of variation in functionally important regions of the human genome is expected to promote understanding of genetically complex diseases. We resequenced 65 candidate genes for CNS disorders in an average of 85 European individuals. The minor allele frequency (MAF), an indicator of weak purifying selection, was lowest in radical amino acid alterations, whereas similar MAF was observed for synonymous variants and conservative amino acid alterations. In noncoding sequences, variants located in CpG islands tended to have a lower MAF than those outside CpG islands. The transition/transversion ratio was increased among both synonymous and conservative variants compared with noncoding variants. Conversely, the transition/transversion ratio was lowest among radical amino acid alterations. Furthermore, among nonsynonymous variants, transversions displayed lower MAF than did transitions. This suggests that transversions are associated with functionally important amino acid alterations. By comparing our data with public SNP databases, we found that variants with lower allele frequency are underrepresented in these databases. Therefore, radical variants obtain distinctively lower database coverage. However, those variants appear to be under weak purifying selection and thus could play a role in the etiology of genetically complex diseases.     

Skeletal abnormalities in the Apert syndrome

This paper reports on skeletal abnormalities in 38 patients with Apert syndrome. Analysis includes alterations in the shoulders, humeri, elbows, hips, knees, rib cage, and spine (except the cervical spine). Some patients had subacromial dimples and elbow dimples during infancy. Mobility at the glenohumeral joint was limited. Progressive limitation in abduction, forward flexion, and external rotation with growth was virtually a constant finding. The acromioclavicular joint was prominent and sometimes had an angular, pointed appearance clinically. This was often associated with atrophic musculature and winging of the scapulae. Limited elbow mobility was common and usually mild in degree. Decreased elbow extension was most often found with decreased flexion, pronation, and supination occurring less frequently. Limited elbow mobility did not change significantly with growth in contrast to the increasing severity observed in the shoulder joint. Short humeri were a constant finding beyond infancy and genua valga of mild degree were present in many cases. Radiographic examination strongly suggests that the Apert syndrome is characterized by a multiple epiphyseal dysplasia. We found delay in appearance of postnatal ossification centers, particularly in the humeral head, greater tuberosity, capitulum, and radial head. Subsequently, these bones became abnormal in shape. Glenoid dysplasia was observed consistently. The neck of the scapula was very short or absent and the inferior margin of the glenoid cavity was poorly demarcated from the infraglenoid tubercle. The humeral head became oblong in shape with relative prominence of the greater tuberosity which compromised abduction. In the elbow, the capitulum was often small and the radial head was flat in many instances. Subluxation or dislocation of the radial head or angulation of the radial neck was observed in some cases. In the hip joint of some adults, the femoral necks were short and broad with prominence of the greater trochanters. Less common radiographic findings are also discussed. © 1993 Wiley-Liss, Inc.