Patent Application
20120190569
The present invention relates to a new nucleic material of the endogenous retroviral genomic type, various nucleotide fragments comprising it or which are obtained from said material, as well as their use as a marker for at least one autoimmune disease or a pathology which is associated with it, a pathological pregnancy or an unsuccessful pregnancy.
The screening of the cDNA library with the aid of the Ppol-MSRV probe (SEQ ID NO: 29) has made it possible to detect overlapping clones allowing the reconstruction of a putative genomic RNA of 7582 nucleotides. —Reconstructed sequence is understood to mean the sequence deduced from the alignment of the overlapping clones—. This genomic RNA has the structure R-U5-gag-pol-env-U3-R. A “blastn” interrogation on several databases, with the aid of the reconstructed genome, shows that a large quantity of related genomic sequences (DNA) exist in the human genome. About 400 sequences have been identified in GenBank (cf
The reconstructed sequence (mRNA) is integrally contained inside the genomic clone RG083M05 (gb A000064) (9.6 kb), and exhibits 96% similarity with two discontinuous regions of this clone which also contains repeat regions at each end. The alignment of the experimental sequences corresponding to the 5′ and 3′ regions of the reconstructed genomic RNA with the DNA of the RG083M05 clone has made it possible to deduce an LTR sequence and to identify elements characteristic of retroviruses, in particular those involved in reverse transcription, namely the PBS (Primer Binding Site) downstream of the 5′ LTR and the PPT (PolyPurine Tract) upstream of the 3′ LTR. It is observed that the U3 element is extremely short in comparison with the mammalian type C retroviruses, and comparable in size to the U3 region generally described in the type D retroviruses and the avian retroviruses. The PBS region is homologous to the PBS of the avian as retroviruses, suggesting the use of the tRNATrp primer for the reverse transcription. Consequently, this new family of HERV is called HERV-W (Human Endogenous RetroVirus).
Phylogenetic analysis in the pol region has shown that the HERV-W family is phylogenetically linked to the ERV-9 and RTVL-H families, and therefore belongs to the family of type I endogenous retroviruses. Phylogenetic analysis of the open reading frame (ORF) of env shows that it is closer to the type D simian retroviruses and the avian reticuloendotheliosis retroviruses than type C mammalian retroviruses, suggesting a C/D chimeric genome structure.
The phylogenetic trees, ‘supported by high “bootstrap” values show that the ERV-9 and HERV-W families are derived from two waves of independent insertions. Thus, the active element(s) at the origin of the HERV-W family is (are) different from that (those) from which the ERV-9 family is derived. Furthermore, the PBS of HERV-W probably uses a tRNATrp whereas ERV-9 probably uses a tRNAArg.
Finally, the members of the HERV-W family are expressed in the placenta, whereas the ERV-9 RNAs are not detected in this tissue.
Biological Functions of HERV-W
The expression of HERV-W restricted to the placenta and the long reading frame potentially encoding a retroviral envelope make it possible to propose physiological biological functions whose impairment could be associated with pathologies.
The expression restricted to the placenta suggests that the expression of retroviral and/or nonretroviral genes under the control of the LTRs may be hormone-dependent. These genes may be adjacent, or under the control of isolated LTRs. A pathology may then result from an aberrant expression following the reactivation of a silent LTR by various factors: viral infection (for example by a member of the Herpesvirus family) or local immune activation. A polymorphism at the level of the LTRs could also promote these events.
The envelope of HERV-W could play a fusogenic role, in particular at the level of cellular subtypes of the placenta. An immunosuppressive peptide of this envelope could protect the fetus against attack by the maternal immune system. Finally, by a mechanism of saturation of receptors, the envelope of HERV-W could play a protective role against exogenous retroviral infections. The impairment of local cellular immunity may result from an immunostimulatory signal carried by the envelope. This effect may be linked to a region carrying a superantigen activity, or to the immunosuppressive region which would become immunostimulatory following either a polymorphism or a dose-effect (overexpression).
Verification of these implications and understanding of the consequences linked to an impairment of the biological functions of the endogenous LTRs or the retroviral envelope may lead to the establishment of methods of diagnosis or of monitoring:
In accordance with the present invention, there has been discovered, in the endogenous state, a new nucleic material, stated explicitly and described below, having the organization of a retrovirus, and capable of being correlated with an autoimmune disease, or a pathology which is associated with it, with a pathological pregnancy or an unsuccessful pregnancy.
The nucleic material according to the present invention, in mRNA form, represents about 8 Kb; it is represented in
The expression “of retroviral type” is understood to mean the characteristic according to which the nucleic material considered comprises one or more nucleotide sequences related to the organization of a retrovirus, and/or to its functional or coding sequences.
This reference nucleic material is related to a human endogenous retrovirus, designated by the expression HERV-W. Consequently, it may be obtained by any appropriate technique for screening any library of human DNA, or of placental cDNA, as shown below, in particular with nucleic primers or probes synthesized so as to hybridize with all or part of SEQ ID NO: 11.
The present invention also relates to any nucleic or peptide product, obtained or derived from the reference nucleic material, according to SEQ ID NO: 11.
And finally, the invention relates to the various correlations which may be made between the above-mentioned nucleic material, and/or its derived products, with any autoimmune disease and/or a pathology which is associated with it, as well as with cases of pathological pregnancy or of unsuccessful pregnancy.
“Autoimmune” is understood to mean in particular:
The present invention relates, first of all, to a nucleic material of the retroviral genomic type, in isolated or purified state, at least partially functional or nonfunctional.
This material is characterized in that its genome comprises a reference nucleotide sequence chosen from the group including the sequences SEQ ID NOs: 1 to 15, their complementary sequences, and their equivalent sequences, in particular the nucleotide sequences exhibiting, for any sequence of 100 contiguous monomers, at least 50% and preferably at least 70%, for example at least 90% homology with respectively said sequences SEQ ID NOs: 1 to 15.
This material is also characterized in that its genome comprises a reference nucleotide sequence, encoding any polypeptide exhibiting, for any contiguous sequence of at least 30 amino acids, at least 50% homology, preferably at least 70% homology, more preferably at least 80% homology, and even more preferably at least 90% homology with a peptide sequence capable of being encoded by at least a functional part of the reference nucleotide sequence as defined above.
In particular, this material comprises a nucleic fragment inserted between two sequences corresponding respectively to the LTR region and to the gag gene for the retroviral genomic structure, in particular a nucleic fragment consisting of or comprising the sequence SEQ ID NO: 12.
The invention also relates to a nucleic material of the subgenomic retroviral type, consisting of a nucleotide sequence identical to SEQ ID NO: 11, with a deletion as exemplified by the clones cl.PH74 (SEQ ID NO: 7), cl.PH7 (SEQ ID NO: 8) and cl.Pi5T (SEQ ID NO: 9), this deletion resulting or otherwise from a splicing strategy.
The above-defined nucleic material comprises at least one functional nucleotide sequence encoding at least one retroviral protein, and/or at least one regulatory nucleotide sequence.
Next, the invention relates to any nucleotide fragment of at least 100 bases, comprising a nucleotide sequence chosen from the group comprising:
a) all the nucleotide sequences, partial and complete, of a nucleic material as defined above
b) all the nucleotide sequences, partial and complete, of a clone chosen from the group including the clones:
c) the sequences which are respectively complementary to the sequences according to a) and b)
d) the sequences which are respectively equivalent to the sequences according to a) to c), in particular the nucleotide sequences exhibiting, for any sequence of 100 contiguous monomers, at least 50%, and preferably at least 70%, or even better at least 80%, for example at least 90% homology with the sequences a) to c).
The invention also relates to any nucleic probe for the detection of a nucleic material, inserted or otherwise into a nucleic acid, characterized in that it is capable of hybridizing specifically with a nucleic material, as defined above.
Such a probe comprises a marker or otherwise.
The invention also relates to a nucleic primer for the amplification by polymerization of an RNA or of a DNA, characterized in that it comprises a nucleotide sequence capable of hybridizing specifically with a nucleic material or a nucleic fragment, as defined above.
By way of example, a nucleic probe or nucleic primer according to the invention is characterized in that it consists of a nucleotide sequence chosen from the group including SEQ ID NOs: 16 to 28.
The invention also relates to any RNA or DNA, and in particular a replication vector, comprising a nucleotide fragment, as defined above.
The invention also relates to any peptide encoded by any open reading frame belonging to a nucleotide fragment, as defined above, in particular polypeptide, for example oligopeptide forming an antigenic determinant recognized by sera from patients affected by an autoimmune disease, or a pathology which is associated with it, or from patients having a pathological pregnancy or an unsuccessful pregnancy.
By way of example, this polypeptide is encoded by a nucleotide fragment comprising an open reading frame encoding one or more retroviral ENV proteins.
Finally, the invention relates to:
The invention also relates to a method for the molecular labeling of an autoimmune disease or of a pathology which is associated with it, of pathological pregnancy or of unsuccessful pregnancy, characterized in that any nucleotide fragment, as defined above, either in RNA form or in DNA form, is identified and/or quantified in any biological body material, in particular body fluid.
By way of example, according to such a method, cells expressing a nucleotide fragment, as defined above, are detected in said biological body material.
The invention relates to a diagnostic and/or therapeutic application of a nucleic material, of a nucleotide fragment or of a peptide defined above, and as such, another subject of the invention is a diagnostic composition or a therapeutic composition comprising said material, said fragment or said peptide.
Before detailing the invention, various terms used in the description and the claims are now defined:
(a) any fragment capable of at least partially hybridizing with the complement of the reference fragment,
(b) any fragment whose alignment with the reference fragment leads to identical contiguous bases being identified in a larger number than with any other fragment obtained from another taxonomic group,
(c) any fragment resulting or capable of resulting from the natural variability within the same individual, and from the natural diversity from one individual to another within the same species, from which it is obtained,
(d) any fragment capable of resulting from genetic engineering techniques applied to the reference fragment,
(e) any fragment, containing at least eight contiguous nucleotides, encoding a peptide homologous or identical to the peptide encoded by the reference fragment,
(f) any fragment different from the reference fragment by insertion, deletion, substitution of at least one monomer, extension, or shortening at least one of its ends; for example, any fragment corresponding to the reference fragment, flanked at least one of its ends by a nucleotide sequence not encoding a polypeptide,
(a) any polypeptide possessing a sequence in which at least one amino acid has been substituted with an analogous amino acid;
(b) any polypeptide having an equivalent peptide sequence obtained by natural or induced variation of said reference polypeptide, and/or of the nucleotide fragment encoding said polypeptide,
(c) a mimotope of said reference polypeptide,
(d) any polypeptide in whose sequence one or more amino acids of the L series are replaced by an amino acid of the D series, and vice versa,
(e) any polypeptide into whose sequence a modification of the side chains of the amino acids has been introduced, such as for example an acetylation of the amine functions, a carboxylation of the thiol functions, an esterification of the carboxyl functions,
(f) any polypeptide in whose sequence one or more peptide bonds have been modified, such as for example the carba, retro, inverse, retro-inverse, reduced and methyleneoxy bonds,
(g) any polypeptide of which at least one antigen is recognized by an antibody directed against a reference polypeptide,
The expressions relating to order which are used in the present description and the claims, such as “first nucleotide sequence” are not selected to express a particular order, but to define the invention more clearly.
Detection of a substance or agent is understood to mean hereinafter both an identification and a quantification, or a separation or isolation of said substance or of said agent.
The invention will be understood more clearly upon reading the detailed description which follows, made with reference to the appended figures in which:
the location of the probes used (Pgag-LB19, Ppro-E, Ppol-MSRV and Penv-C15);
the splice donor sites [DS1 (SEQ ID NOs: 36 and 38) and DS2 (SEQ ID NO: 39)] and acceptor sites [AS1 (SEQ ID NOs: 37 and 40), AS2 (SEQ ID NO: 41) and AS3 (SEQ ID NO: 42)];
the sequences obtained from the clone RG083M05, in the lower-case boxes, and the sequences derived from experimental placental clones (mRNA), in the upper-case boxes;
the putative ORFs (ORF1, ORF2 and ORF3); and
an insert of 2 Kb present in DNA form but not detected in RNA form, represented in the form of vertical hatches.
The other conventions used in this figure are the same as those for
the percentage similarity with respect to the reconstructed genomic RNA (Recons RNA);
the presence of repeat sequences at each end of these genomes (repeats); and
the presence and the size of the open reading frames (ORFs).
The nucleic material previously presented explicitly was discovered and characterized at the end of the experimental protocol described below, it being understood that this protocol cannot limit the scope of the present invention and of the accompanying claims.
The information relating to the organization of HERV-W were obtained by testing a placental cDNA library (Clontech cat#HL5014a) with the probes Ppol-MSRV (SEQ ID NO: 29) and Penv-C15 (SEQ ID NO: 31) (cf Example 8), and then performing a “gene walking” technique with the aid of the new sequences obtained. The experiments were carried out with reference to the recommendations of the supplier of the library. PCR amplifications on DNA were also exploited in order to understand this organization.
A number of clones were selected and sequenced, cf
With the aid of these clones, by carrying out sequence alignments, a model of complete sequence of HERV-W was produced. The spliced RNAs were identified as well as the potential splice donor and acceptor sites. This set of information is shown in
The putative genetic organization of HERV-W in RNA form is the following (SEQ ID NO: 11):
gene 1.7582
location of the clones on the reconstructed genomic RNA sequence
A “blastn” interrogation of several databases, with the aid of the reconstructed genome, shows that a large quantity of related sequences exist in the human genome. About 400 sequences were identified in GenBank and more than 200 sequences in the EST library, and the majority as antisense. The 4 sequences most significant in size and in similarity, illustrated in
the human clone RG083M05 (gb AC000064) whose chromosomal location is 7q21-7q22,
the human clone BAC378 (gb U85196, gb AE000660) corresponding to the alpha delta locus of the T cell receptor, located in 14q11-12,
the human cosmid Q11M15 (gb AF045450) corresponding to the 21q22.3 region of chromosome 21,
the cosmid U134E6 (embl Z83850) on chromosome Xq22.
The location of the aligned regions for each of the clones is indicated and the affiliation to a chromosome is indicated in square brackets. The percentage similarity (without broad deletions) between the 4 sequences and the reconstructed genomic RNA is indicated, as well as the presence of repeat sequences at each end of the genome and the size of the largest reading frames (ORF). Repeat sequences are found at the ends of 3 of these clones. The reconstructed sequence is integrally contained inside the clone RG083M05 (9.6 Kb) and exhibits a 96% similarity. However, the clone RG083M05 exhibits an insert of 2 Kb situated immediately downstream of the untranslated 5′ region (5′ UTR). This insert is also found in two other genomic clones which exhibit a deletion of 2.3 Kb immediately upstream of the untranslated 3′ region (3′ UTR). No clone contains the three functional reading frames (ORFs) gag, pol and env. The clone RG083M05 shows an ORF of 538 amino acids (AA) corresponding to a whole envelope. The cosmid Q11M15 contains two large contiguous ORFs of 413 AA (frame 0) and 305 AA (frame +1) corresponding to a truncated pol polyprotein.
A phylogenetic analysis was carried out at the level of the nucleic acids on 11 different subregions of the reconstructed genomic RNA, and at the protein level on 2 different subregions of env. All the trees obtained exhibit the same topology regardless of the region studied. This is illustrated in
Comparison at the protein level between the most conserved regions of the retroviral env proteins shows that the HERV-W family is closer to the type D simian retroviruses and the avian reticuloendotheliosis retroviruses than the type C mammalian retroviruses.
This suggests a C/D chimeric genomic structure.
The reconstructed sequence (RNA) is integrally contained inside the genomic clone RG083M05 (9.6 Kb) and exhibits a 96% similarity with two discontinuous regions of this clone which also contains repeat regions at each end. The alignment of the experimental sequences corresponding to the 5′ and 3′ regions of the genomic RNA reconstructed with the DNA of the clone RG083M05 [5′(5-RG-28000-28872) (SEQ ID NO: 43) and 3′(3-RG-3750038314) (SEQ ID NO: 44)] made it possible to deduce an LTR sequence and to identify elements characteristic of the retroviruses, in particular those involved in the reverse transcription, namely PBS downstream of the 5′ LTR and the PPT upstream of the 3′ LTR (cf
The PBS region is homologous to the PBS of the avian retroviruses.
Organization in DNA Form
PCR amplifications were carried out on whole HERV-W clones recovered on human genomic library (see Example 1 for the mode of production), using the following oligonucleotide pairs:
The PCRs were carried out under the following conditions:
oligonucleotides at the concentration of 0.33 microMolar
TAQ polymerase buffer Boerhinger 1×
0.5 unit of TAQ polymerase Boerhinger
mixture of dNTP at 0.25 mM each
0.5 mg of human DNA
final volume 100 ml
PCR conditions (95° C., 5 min)×1, (95° C., 30 sec+54° C., 30 sec+72° C. 3 min)×35.
The PCR products were then deposited on 1% agarose gel to be analyzed after migration. The set of PCRs gives amplification fragments of the expected size, except for the LTR-4991--gag-4619 PCR which gives a fragment of size greater by about 2 Kb relative to the expected size (deduced from cDNAs from the placental library). The reconstruction of HERV-W in endogenous DNA form therefore represents an entity of about 10 Kb.
After cloning, sequencing and analysis of the PCR-4992, gag-4619, the presence of a region of insertion is observed between LTR and gag of SEQ ID NO: 12 (clone cl.6A5). This region does not correspond to an untranslated traditional region of a retrovirus: no y or PBS region.
The products of PCR poi-3390, poi-5144 were also cloned and two of the clones obtained were sequenced. The result of these sequences is given by the clones cl.7A20 (SEQ ID NO: 13) and cl.7A21 (SEQ ID NO: 14). Comparison of these two nucleotide sequences gives a score of 90% homology for the relevant region, thus showing the variability of HERV-W in the same individual.
HERV-W in DNA form is proposed in
General organization: transcription process
The various cDNA clones having been obtained, results acquired in PCR on DNA, there is deduced:
The result of PCR on DNA showing the presence of an insert of 2 Kb between the LTR and gag regions suggests that the cDNAs isolated from the placenta are obtained from the expression of a genome of the RG083M05 type.
The probes gag (Pgag-LB19, SEQ ID NO: 30) and protease (Ppro-E, SEQ ID NO: 32) reveal an RNA having a size close to 8 Kb, the probe Penv-C15 (SEQ ID NO: 31) reveals, in addition, an RNA close to 3.1 Kb. Two probes defined in the untranslated 5′ region, obtained by screening of the cDNA library reported above (probe P5′-gag-cl.6A2 derived from the clone cl.6A2 and probe P5′-env-cl.24.4 derived from the clone cl.24.4) reveal the preceding two RNAs and an RNA of about 1.3 Kb. This distribution of the RNAs is typical of complex retrovirus transcripts: a genomic RNA encoding gag-pro-pol, a subgenomic RNA encoding the envelope, and one or more multispliced RNAs potentially encoding regulatory genes.
The half-life of such an RNA (LTR-R-U5Insertion-GAG-POL-ENV-U3-R-HERV-W) is probably very short, because no RNA of 10 Kb is detected in Northern blotting. By analyzing and comparing sequences, the potential splice donor sites (DS1 and DS2) and acceptor sites were defined and described in
Various healthy human tissues were tested by the Northern-blot technique (Human Multiple Tissue Northern Blot, Clontech cat#7760-1), with the aid of the probes Ppol-MSRV (SEQ ID NO: 29), Pgag-LB19 (SEQ ID NO: 30), Penv-C15 (SEQ ID NO: 31), Ppro-E (SEQ ID NO: 32), P5′-gag-cl.6A2 and P5′-env-cl.24.4, labeled as described in Example 1. The experiments were carried out following the recommendations of the manufacturers, and the autoradiographs were exposed for 5 days. Analysis of the results reveals transcription products only in the placenta, and in none of the other human tissues tested (heart, brain, lungs, liver, skeletal muscle, kidney and pancreas).
Using an RNA Dot-Blot technique (Clontech: Human RNA Master Blot Cat#7770-1), and using the experimental protocol recommended by the manufacturer, about forty other tissues, including fetal tissues, were tested: only the placenta gives a specific response after hybridization with the probes' Pgag-LB19 (SEQ ID NO: 30) and Penv-C15 (SEQ ID NO: 31).
It is observed that a signal is observed in the kidney in RNA Dot-Blot, which is infirmed by the Northern-blot analysis.
The screening of a placental cDNA library with the aid of a probe defined in the untranslated 5′ region made it possible to isolate a cDNA defined by an untranslated 5′ region (5′ NTR), a splicing junction, a coding sequence, an untranslated 3′ region (3′ NTR) and a polyadenylated tail, cl.PH74 (SEQ ID NO: 7). This clone corresponds to a spliced RNA encoding an envelope. By comparing sequences between this cDNA and the endogenous HERV-W model proposed according to
The identification of this joining region makes it possible to establish a method of discriminating between endogenous retroviral RNA and DNA, using, in a PCR, an oligonucleotide defined on this joining region, in particular an oligonucleotide chosen from the env gene (Oligo 4986, according to SEQ ID NO: 25).
The PCRs were carried out under the following conditions:
oligonucleotides at the concentration of 0.33 microMolar
TAQ polymerase buffer Boerhinger 1×
0.5 unit of TAQ polymerase Boerhinger
mixture of dNTP at 0.25 mM each
0.5 mg of human DNA
final volume 100 ml
On 10 different DNAs tested, this type of PCR did not make it possible to obtain amplification products. On the other hand, on cDNA derived from placental RNA or from cells expressing HERV-W, this PCR gives an amplification product. This result therefore confirms the specifically RNA nature of this subgenomic fragment.
The splicing strategy described in Example 5 is compatible with the presence of three reading frames ORF1 (SEQ ID NO: 33), ORF2 (SEQ ID NO: 34) and ORF3 (SEQ ID NO: 35) (cf
The screening of a placental cDNA library made it possible to isolate a cDNA (SEQ ID NO: 7, cl.PH74) defined by an untranslated 5′ region (5′ NTR), a splicing junction, a coding sequence, an untranslated 3′ region (3′ NTR) and a polyadenylated tail. The coding sequence is 538 amino acids (SEQ ID NO: 33). The analyses carried out on databanks make it possible to identify characteristics of a complete retroviral envelope: initiation of translation of an envelope polyprotein, of a highly hydrophobic leader peptide of about 21.amino acids, of a surface protein SU, of a transmembrane protein TM. These two protein entities exhibit different potential glycosylation sites. An immunosuppressive region is identified within the TM protein.
22 bp and 95 bp upstream of the splice acceptor site, two initiation codons were respectively found which were capable of directing the synthesis of 52 AA (ORF2, SEQ ID NO: 34) and of 48 AA (ORF3, SEQ ID NO: 35). ORF2 consists of part of the carboxyterminal end of env and ORF3 corresponds to a different but overlapping translation.
No significant homology was found by “blast” interrogation. However, an LFASTA interrogation in a sub-databank limited to the Retroviridae, ORF2 and ORF3 showed a percentage identity of 35% with, respectively, Rex of the human and primate lymphotropic T virus, and with Tat of the simian immunodeficiency virus.
The number of copies present in the human genome of each of the sequences is evaluated by a DotBlot technique, with the aid of the probes Pgag-LB19 (SEQ ID NO: 30), Ppro-E (SEQ ID NO: 32) and Penv-C15 (SEQ ID NO: 31)
Each of the probes is denatured and deposited on a Hybond N+ membrane in an amount of 2.5, 5, 10, 25, 50, 100 pg per deposit. 0.5 mg of human DNA are also deposited on the same membrane. The membranes are dried for 2 hours under vacuum at 80° C. The membranes are then hybridized with the deposited probe. The techniques for labeling the probes, for hybridization and for washing the membranes are the same as for the Southern blotting. After autoradiography of the membranes, levels of signal intensity which are proportional to the deposits on the membrane are observed. After cutting out the hybridization zones, scintillation counting is carried out. By comparison between the dilution series for the probe deposited on the membrane and the result obtained with the human DNA, it is possible to evaluate the number of copies per haploid genome of each of the regions covered by the probes:
The screening of 106 clones of a human placental DNA library (Clontech cat* H15014b) made it possible to count 144 clones recognized by the probe Pgag-LB19, and 64 clones recognized by the probe Penv-C15. 13 clones hybridized conjointly with the probes Penv-C15 and Pgag-LB19 were isolated, confirming the presence of several copies of a genome possessing both gag and env, without consideration of functionality.
The nucleic material, the nucleotide sequences and the peptides or proteins which may be expressed by said materials and sequences may be used to detect, predict, treat and monitor any autoimmune disease, and the pathologies which are associated with it, as well as in cases of pathological pregnancy or of unsuccessful pregnancy.
Indeed, the objective and experimental data make it possible to link retrovirus and autoimmune diseases and retrovirus and pregnancy disorders:
(1) common mechanisms are used in the retroviral pathologies and in autoimmune diseases (presence of autoantibodies, of immune complexes, cellular infiltration of certain tissues, neurological disorders).
(2) pathological disorders comparable to certain autoimmune diseases appear during infections with HIV and HTLV retroviruses (Sjogren syndrome, disseminated lupus erythematosus, rheumatoid arthritis and the like).
(3) a reverse transcriptase activity was detected and retroviral-type particles were observed in the cell culture supernatants of patients suffering from multiple sclerosis (Perron et al., Res. Virol. 1989; 140: 551-561/Lancet 1991; 337: 862-863/Res. Virol. 1992; 143: 337-350) or from rheumatoid arthritis.
(4) autoimmune or chronic inflammatory animal pathologies are linked to endogenous retroviruses; some of them are used as animal models of human diseases (insulin-dependent diabetes, disseminated lupus erythematosus).
(5) significant levels of endogenous anti-retrovirus antibodies have been described in the context of autoimmune, systemic or inflammatory diseases; other data of this nature were communicated by several authors at the IVth European meeting on endogenous retroviruses (Uppsala, October 1996). According to Venables (communiques of the IVth European meeting on endogenous retroviruses, Uppsala, October 1996), a significantly high level of antiHERV-H antibodies are found during pregnancy but also in the context of various autoimmune disorders such as Sjogren syndrome, disseminated lupus erythematosus or rheumatoid ‘arthritis, without, however, any proof of its direct involvement being provided up until now.
The involvement of the retroviruses in the autoimmune phenomenon remains compatible with the multifactorial character of the autoimmune, systemic or inflammatory diseases which confront genetic, hormonal, environmental and infectious factors.
The particles observed in the cell culture supernatants from patients suffering from multiple sclerosis (Perron et al., Res. Virol. 1989; 140: 551-561/Lancet 1991; 337: 862-863/Res. Virol. 1992; 143: 337-350) or from rheumatoid arthritis (unpublished data) may result from the expression: (i) of an endogenous retrovirus competent for replication, (ii) of several defective endogenous retroviruses cooperating by a phenomenon of transcomplementation or (iii) of an exogenous retrovirus.
All these observations make it possible to use and consider the above-described biological material as marker for an autoimmune disease or for pregnancy disorders.
In particular, the following labeling techniques are considered:
| Number | Date | Country | Kind |
|---|---|---|---|
| 97/08815 | Jul 1997 | FR | national |
This is a divisional of application Ser. No. 10/717,580 filed Nov. 21, 2003, which is a continuation of application Ser. No. 09/446,024 filed Dec. 16, 1999, now abandoned, which is a National Stage Application of PCT/FR98/01442 filed Jul. 6, 1998, and claims the benefit of French Application No. 97/08815 filed Jul. 7, 1997. The entire disclosures of the prior applications are hereby incorporated by reference herein in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10717580 | Nov 2003 | US |
| Child | 13336712 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 09446024 | Dec 1999 | US |
| Child | 10717580 | US |
