Antibodies against a human endogenous retrovirus and the preponderance of env splice variants in multiple sclerosis patients.

The human endogenous retrovirus HERV-H is associated with multiple sclerosis (MS). Previously performed reverse transcriptase-polymerase chain reactions (RT-PCR) on virion-RNA demonstrated sequence variants of the HERV-H family located in the particulate fraction of MS patient plasma samples and not in controls. In this study a significantly elevated level of antibodies towards peptides derived from HERV-H/RGH-2 DNA sequences in serum and cerebrospinal fluid (CSF) from MS patients is demonstrated. Further, Wistar rats immunized with purified virions develop a specific serologic response, indicating that some virion proteins are encoded by HERV-H-related sequences. Also shown is that in RNA from blood cells, a HERV-H protease-env splice variant can be found together with an env splice variant in about 40% of MS patients but only in 10% of controls. The results substantiate the association between activated HERV-H and MS, but a causal relationship is yet to be demonstrated. HERV-H could represent a causal factor either by eliciting an autoimmune response or through the pathogenic potential of the retrovirus itself.

[Endogenous retroviruses in multiple sclerosis]. / Undersøgelse af endogene retrovirus i multipel sklerose.

INTRODUCTION: In recent years, it has been suggested that human endogenous retroviruses (HERVs) may play a role in autoimmune diseases. HERVs represent both putative susceptibility genes and putative pathogenic viruses in multiple sclerosis (MS). Initially, our objective was to characterize a retrovirus produced by MS derived cell lines and to investigate this association in vivo. METHODS: The retrovirus was identified by RT-PCR on virion RNA purified from RT-positive retroviral particles from the MS cell lines, and from blood samples from MS patients. Wistar rats were immunized with purified virions and serological responses analyzed by ELISA. RESULTS: Sequence variants highly homologous to the HERV-H family were found. The same sequences were specifically found in the particulate fraction of a series of MS patient plasma samples and were absent in controls. A database search demonstrated HERV-H copies in several chromosome regions implied in MS susceptibility. Virion-immunized rats developed a specific serological response towards HERV-H peptides indicating that immunogenic virion proteins are encoded by HERV-H. DISCUSSION: Activation of normally replicatively quiescent HERVs may be causally involved in MS.

Characterization of retroviruses from patients with multiple sclerosis.

These studies were performed to characterize retroviruses found in cell lines spontaneously developed from peripheral blood mononuclear cells (PBMNC) from 6 multiple sclerosis patients, a patient with progressive myelopathy and a healthy control. The cell lines are B-lymphoblastoid and produce Epstein-Barr virus (EBV) particles or express EBV proteins. The B-lymphoblastoid cell lines are also characterized by production of low, fluctuating amounts of retrovirus. The low productivity complicates purification and characterization, but implementation of product-enhanced reverse transcriptase (PERT) assays has provided a highly useful tool for monitoring retrovirus production. By electron microscopy, the retroviral particles appear type-C-like. Functional assays indicate the presence of Pol, Gag and Env. Indirect ELISA demonstrates a significant relation between disease activity and reactivity towards retroviral peptides. Molecular characterization is primarily based on RT-PCR, cloning, sequencing and Northern- or Southern analyses. Molecular characterization is continuing.

Transcription of human 5S rRNA genes is influenced by an upstream DNA sequence.

Six human 5S rRNA genes and gene variants and one pseudogene have been sequenced. The six genes/variants were transcribed in a HeLa cell extract with about equal efficiency. Three genes contain the Sp1 binding sequence GGGCGG in position -43 to -38 and three genes contain the Sp1 like sequence GGGCCG in this position. The six genes contain furthermore one Sp1 binding site in a position about -245 and one ATF recognition site in a position about -202. A 12 bp sequence (GGCTCTTGGGGC) found in position -32 to -21 strongly influenced the transcriptional efficiency in vitro. This 12-mer, designated the D box, has also been found upstream a 5S rRNA gene from hamster and mouse. Removal of the Sp1 binding sites had no effect on the transcription in vitro whereas the transcriptional efficiency decreased to 10% if the D box was removed from the human 5S rRNA gene.

Salmonella identification by the polymerase chain reaction.

Polymerase chain reaction (PCR) primers for genus specific detection of Salmonella have been selected from a Salmonella-specific fragment of 2.3 kilobases (kb). Due to interserovar sequence diversity within this fragment, primer selection was based on DNA sequence alignment of sequences from 20 different Salmonella serovars. The specific PCR product of 429 base pairs (bp) was formed from 144 of 146 salmonella strains tested (116 of 118 serovars). The two false-negative strains belonged to two different serovars of the rarely isolated subspecies IIIa (monophasic S. arizonae). No product was produced in any of 86 non-Salmonella Enterobacteriacea strains tested, covering 41 species from 21 genera.

Characterization of human 5S rRNA genes.

The human 5S rRNA genes are found in clusters of tandem repeated units. We have cloned and partially characterized six restriction fragments from two clusters of 2.3 kb and 1.6 kb repeats, respectively. Four fragments from the cluster of 2.3 kb repeats contain a 5S rRNA gene and one fragment contains a gene variant with an additional nucleotide in the internal control region. A fragment from the 1.6 kb cluster contains a gene and is highly homologous to the 2.3 kb repeats, except for a large deletion in the 3'-flanking region starting 12 bp downstream of the gene. The number of genes and closely related gene variants is found to be 300-400 per haploid human genome. 100-150 of these are found in 2.3 kb repeats and 5-10 are found in 1.6 kb repeats. The total number of 5S rRNA sequences, including pseudogenes, is 1700-2000 per haploid genome. The genes and the gene variant are transcribed equally efficient in a HeLa cell extract. If 5'-flanking sequences, including a GC-motif in the -40 region, are removed from the genes, transcription is reduced with a factor 10 or more, suggesting that sequences upstream of the coding region are important for the level of transcription.

Fine mapping of human 5S rRNA genes to chromosome 1q42.11----q42.13.

The human 5S rRNA genes have been localized by in situ hybridization to metaphase chromosomes. Tritiated RNA probes were made by transcription from 2.300-bp and 638-bp DNA fragments containing an isolated human 5S rRNA gene. Hybridization to metaphase spreads from a balanced reciprocal translocation carrier, 46,XX.t(1:7)(q42.13;p11.1), showed that the 5S rRNA genes were entirely localized on the normal and the derivative chromosome 1. This narrows the chromosome position of the major fraction of 5S rRNA genes and pseudogenes to the region 1q42.11----q42.13.

Optimization of in situ hybridization to human metaphase chromosomes.

A simplified method describing optimal conditions for in situ hybridization to human chromosomes is presented. A 1.5-kb DNA fragment coding for part of the 28 S rRNA was subcloned into pSP65. Tritium-labeled RNA was synthesized as runoff transcripts and 39-67% of the labeled probes specifically hybridized to the nucleolar organizer regions of the acrocentric chromosomes. Denaturation performed with 70% formamide, 1 mM EDTA, 2 X SSC gave a high specific hybridization with both fresh chromosome spreads (1-8 weeks) and older preparations (3-6 months). To obtain good chromosome morphology and a high specific hybridization it was important to neutralize the final formamide denaturation mixture containing 70% formamide, 1 mM EDTA, and 2 X SSC, whereas it was unimportant to deionize the formamide. Freshly made slides denatured with 0.15 M NaOH in 70% ethanol hybridized equally well with the rRNA probe. Despite treatment of the chromosomes with RNases before denaturation the following proteinase K and the acetylation steps recommended could be omitted without degradation of the rRNA probe as judged from the high specific hybridization to the nucleolar organizer regions.

Constituents of Erysimum inconspicuum. Two sulfur-containing lactone compounds.

The alcohol extract of Erysimum inconspicuum fruits, which exhibited cytotoxic activity against the KB cell line and some activity against the P-388 lymphocytic leukemia in vivo, was studied. Strophanthidin, uzarigenin, and two sulfur-containing lactones, erysulfone[6-methylsulfonyl-4-hydroxyhexanoic acid lactone] and erysulfoxide[6-methylsulfinyl-4-hydroxyhexanoic acid lactone], were isolated and characterized by spectral data.