Detection of Simian Immunodeficiency Virus in Semen, Urethra, and Male Reproductive Organs during Efficient Highly Active Antiretroviral Therapy.

UNLABELLED: A number of men receiving prolonged suppressive highly active antiretroviral therapy (HAART) still shed human immunodeficiency virus (HIV) in semen. To investigate whether this seminal shedding may be due to poor drug penetration and/or viral production by long-lived cells within male genital tissues, we analyzed semen and reproductive tissues from macaques chronically infected with simian immunodeficiency virus mac251 (SIVmac251) who were treated for 4 months with HAART, which was intensified over the last 7 weeks with an integrase inhibitor. We showed that a subset of treated animals continued shedding SIV in semen despite efficient HAART. This shedding was not associated with low antiretroviral drug concentrations in semen or in testis, epididymis, seminal vesicles, and prostate. HAART had no significant impact on SIV RNA in the urethra, whereas it drastically reduced SIV RNA levels in the prostate and vas deferens and to a lesser extent in the epididymis and seminal vesicle. The only detectable SIV RNA-positive cells within the male genital tract after HAART were urethral macrophages. SIV DNA levels in genital tissues were not decreased by HAART, suggesting the presence throughout the male genital tract of nonproductively infected cells. In conclusion, our results demonstrate that 4 months of HAART induced variable and limited control of viral infection in the male reproductive organs, particularly in the urethra, and suggest that infected long-lived cells in the male genital tract may be involved in persistent seminal shedding during HAART. These results pave the way for further investigations of male genital organ infection in long-term-treated infected individuals. IMPORTANCE: A substantial subset of men receiving prolonged HAART suppressing viral loads in the blood still harbor HIV in semen, and cases of sexual transmission have been reported. To understand the origin of this persistence, we analyzed the semen and male reproductive tissues from SIV-infected macaques treated with HAART. We demonstrated that persistent seminal shedding was not linked to poor drug penetration in semen or semen-producing prostate, seminal vesicle, epididymis, and testis. We revealed that HAART decreased SIV RNA to various extents in all male genital organs, with the exception of the urethra, in which SIV RNA(+) macrophages were observed despite HAART. Importantly, HAART did not impact SIV DNA levels in the male genital organs. These results suggest that infection of male genital organs, and particularly the urethra, could be involved in the release of virus in semen during HAART.

[Validation of intensity modulated radiation therapy patient plans with portal images]. / Validation des plans de radiothérapie conformationnelle avec modulation d'intensité avec les images portales.

The goal of this study was to show the feasibility of step and shoot intensity-modulated radiation therapy pre-treatment quality control for patients using the electronic portal imaging device (iViewGT) fitted on a Sli+ linac (Elekta Oncology Systems, Crawley, UK) instead of radiographic films. Since the beginning of intensity-modulated radiation therapy treatments, the dosimetric quality control necessary before treating each new patient has been a time-consuming and therefore costly obligation. In order to fully develop this technique, it seems absolutely essential to reduce the cost of these controls, especially the linac time. Up to now, verification of the relative dosimetry field by field has been achieved by acquiring radiographic films in the isocenter plane and comparing them to the results of the XiO planning system (Computerized Medical Systems, Missouri, USA) using RIT113 v4.1 software (Radiological Imaging Technology, Colorado, USA). A qualitative and quantitative evaluation was realised for every field of every patient. A quick and simple procedure was put into place to be able to make the same verifications using portal images. This new technique is not a modification of the overall methodology of analysis. The results achieved by comparing the measurement with the electronic portal imaging device and the calculation with the treatment planning system were in line with those achieved with the films for all indicators we studied (isodoses, horizontal and vertical dose profiles and gamma index).

Protection of swine against post-weaning multisystemic wasting syndrome (PMWS) by porcine circovirus type 2 (PCV2) proteins.

Porcine circovirus type 2 (PCV2) is known to be associated with post-weaning multisystemic wasting syndrome (PMWS), a recently described disease of young pigs. Since no PCV2 vaccine was available so far, we have developed a specific PCV2 vaccine candidate. The Orf1-encoded replication protein and Orf2-encoded capsid protein of PCV2 were expressed and detected in either mammalian or insect expression systems. In a first trial, Orf2 protein was found to be a major immunogen, inducing protection in a prime-boost protocol; the piglets received a first injection with plasmids directing Orf2 protein and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression, followed by a second injection, a fortnight later, associated with baculovirus-expressed Orf2 protein. As evaluated by growth parameters, clinical signs (fever), seroconversion, the pigs were protected against a PCV2 challenge after vaccination. In a second trial, protection induced by a subunit vaccine was even better than the one induced by DNA vaccine, since PCV2 replication was completely inhibited.

An ORF2 protein-based ELISA for porcine circovirus type 2 antibodies in post-weaning multisystemic wasting syndrome.

Porcine circovirus type 2 (PCV2) plays a crucial role in the pathogenesis of post-weaning multisystemic wasting syndrome (PMWS) in swine. As PCV2 displays significant homology with PCV1 (a non-pathogenic virus) at the nucleotide and amino-acid level, a discriminative antigen is needed for specific serological diagnosis. The ORF2-encoded capsid protein from PCV2 was used to develop an indirect enzyme-linked immunosorbent assay (ELISA). GST-fused capsid protein from PCV2 and GST alone (both expressed in recombinant baculovirus-infected cells) were used as antigens for serodiagnosis. The specificity of the ELISA for detection of PCV2 antibodies was demonstrated in sera from pigs experimentally infected with PCV1, PCV2 and other swine viruses. The semi-quantitative nature of the test was evaluated versus an immunoperoxidase monolayer assay (IPMA). The ELISA was performed on 322 sera from pigs in eight Brittany herds and compared with IPMA. The sensitivity (98.2%) and specificity (94.5%) of this test were considered suitable for individual serological detection. High PCV2 seroprevalence was found in sows and pigs at the end of the growth phase (18-19 weeks) in all eight herds. The seroprevalence in piglets (11-17 weeks) was statistically correlated with clinical symptoms of PMWS (93% in affected versus 54%, in non-affected farms). A cohort study performed in PMWS-free farms showed that 57% of piglets exhibited active seroconversion after 13 weeks, indicating that PCV2 infection occurred earlier in PMWS-affected piglets.

Identification and IFNgamma-regulation of differentially expressed mRNAs in murine microglial and CNS-associated macrophage subpopulations.

CNS-resident macrophages (microglia and CNS-associated macrophages) are the main immunocompetent cells of the central nervous system (CNS) and respond by rapid activation to brain injury. Molecular events occurring during IFNgamma-activation and identification of potential markers of the CNS-resident macrophage subsets were investigated using microglial-derived clones (EOC) differing in their morphology and their antigen presenting activities for CD4+ and CD8+ T-cells. By applying the subtractive process of cDNA representational difference analysis (cRDA), 16 differentially expressed mRNAs were isolated and sequenced, revealing 8 known and 8 novel molecules; 15 of these messages were unpreviously reported in microglia. Two markers of all activated microglial EOC cells were identified (iNOS; IRG-1) and specific subpopulation markers were highlighted, including molecules known to be closely expressed in perivascular spaces. Moreover, some messages could support the distinct morphology, adhesive characteristics, and potential functions of the different clones.

Identification of an immunorelevant ORF2 epitope from porcine circovirus type 2 as a serological marker for experimental and natural infection.

Post-weaning multisystemic wasting syndrome (PMWS) is a recently identified disease of pigs linked to the emergence of a new porcine circovirus (PCV2). We report here the characterization of immunorelevant linear B-cell epitopes of the Open Reading Frame 2-encoded protein (Orf2) from PCV2 by an enzyme-linked immunosorbent assay (ELISA) using experimental antisera collected from pigs inoculated with a PCV2 isolate. Two epitopes spanning residues 69 to 83 and 117 to 131 were specific to PCV2. Antibodies to the 117 to 131 epitope (B- 133) were detected in 22% and 100% of specific pathogen-free (SPF) pig sera 6 and 11 weeks post inoculation, respectively. Cross-sectional studies performed with field sera collected from PMWS-affected herds showed B-133 antibodies in 5% of 8 to 10 week-old pigs, 38% of 13-14 week-old pigs, 62% of 16 to 19 week-old pigs, 56% of 20 to 25 week-old pigs and 45% of 26 to 31 week-old pigs. All these data suggest that epitope B- 133 is a serological marker of PCV2 infection that could be used for the detection of PCV2 antibody response.

An experimental model for post-weaning multisystemic wasting syndrome (PMWS) in growing piglets.

Post-weaning multisystemic wasting syndrome (PMWS) is a comparatively new disease of swine, and known to occur in France since 1996. A porcine circovirus type 2 (PCV2) is found in the lesions of affected piglets. Six piglets aged 10-13 weeks were obtained from a French PMWS-affected farm. Two showing characteristic signs of PMWS (palor, weakness and emaciation) remained in poor condition and were finally killed 6 and 9 days after their arrival in the experimental unit. Tissue homogenates from these two piglets were used to reproduce mild PMWS in specific pathogen-free (SPF) piglets. This mild PMWS consisted of pyrexia (up to 41.7 degrees C) and growth retardation (up to 30% of weight reduction compared with controls) commencing 1 week after infection and lasting 3 weeks. In seven additional trials, pyrexia, growth retardation and lesions characteristic of PMWS were consistently produced in SPF and conventional piglets. However, only four of 55 inoculated SPF piglets (7.2%) showed severe wasting disease. One died and the others had to be killed 3 to 4 weeks after inoculation. None of the inoculated animals developed antibodies to any common swine viruses or bacteria, but clear evidence of PCV2 seroconversion was obtained. Our results therefore strongly suggest that PCV2 is the primary aetiological agent of PMWS.

Spatiotemporal regulation of hnRNP M and 2H9 gene expression during mouse embryonic development.

Using the HeLa cell model along with an in vitro splicing system, we have previously shown that hnRNP M and 2H9 are involved in the pre-mRNA splicing process and most interestingly also in heat shock-induced transient splicing arrest by transiently leaving the hnRNP complexes. Due to this unique regulatory function in a mechanism that turns splicing on and off, these two hnRNPs appear as important proteins for controlling gene expression. Here we investigated by in situ hybridization and immunohistochemical staining techniques the expression level of specific mRNA and protein during mouse embryonic development. HnRNP M and 2H9 are found to be expressed at all examined stages (6.5-18.5 days post-coïtum), in a differential manner, and at various levels depending on tissues, cell types and also embryonic stages; fairly high levels of both hnRNPs are always observed in the central nervous system. Furthermore, levels of colocalizing protein and transcript are not always present in the same proportion, thus suggesting a post-transcriptional regulation of hnRNP M and 2H9 gene expression. The complex spatiotemporal variations we observed might well anticipate a role for these two hnRNPs also in modulating splicing, thereby influencing gene expression and further many physiological processes.

The nine C-terminal amino acids of the major capsid protein of the human papillomavirus type 16 are essential for DNA binding and gene transfer capacity.

Four C-terminal deletion mutants of the human papillomavirus 16 L1 protein were expressed in the baculovirus expression system. They consist of the deletion of amino acids 497-505, 477-505, 403-505 and 302-505 (delta C9, delta C31, delta C103 and delta C204 respectively). Only two of the C-terminally deleted proteins, delta C9 and delta C31, retained the ability to form virus-like particles (VLPs) resembling those obtained with the full length L1 protein. Analysis of deleted L1 proteins and corresponding VLPs indicated that the C-terminus was necessary both for DNA binding and DNA packaging. These results were corroborated by the loss of the gene transfer capacities of C-terminal deleted VLPs.

Differential recognition of ORF2 protein from type 1 and type 2 porcine circoviruses and identification of immunorelevant epitopes.

Two types of porcine circovirus (PCV) have been isolated and are referred to as PCV1 and PCV2. PCV1 represents an apathogenic virus, whereas PCV2 is associated with post-weaning multisystemic wasting syndrome. The two PCVs are related, since they display about 70% identity based on nucleotide sequences. In order to discriminate between common and type-specific antigens, an immunocytological approach was used following transfections with cloned circovirus DNAs, as well as recombinant proteins expressed by either baculovirus or plasmid vectors. The ORF1-encoded proteins in the two viruses were shown to be antigenically related, whereas the ORF2 proteins were recognized differentially by polyclonal anti-PCV2 antibodies. Furthermore, PEPSCAN analysis performed on overlapping fragments of the genes encoding part of ORF1 and the entire ORF2 and ORF3 led to the identification of five dominant immunoreactive areas, one located on ORF1 and four on ORF2. However, only some ORF2 peptides proved to be immunorelevant epitopes for virus type discrimination. The potential use of ORF2-derived antigens as diagnostic tools is demonstrated.

Production of recombinant virus-like particles from human papillomavirus types 6 and 11, and study of serological reactivities between HPV 6, 11, 16 and 45 by ELISA: implications for papillomavirus prevention and detection.

The L1 major capsid proteins of human papillomaviruses types 6 and 11 were expressed in insect cells using recombinant baculoviruses. These L1 proteins were shown to self-assemble into virus-like particles resembling papillomavirus virions as previously observed for HPV 16 and 45. However, we observed variations in the yield of virus-like particles among the four genotypes investigated. This suggests that more than one strain of each genotype has to be investigated to obtain the high level of virus-like particle production necessary to develop HPV vaccines or serological tests. Cross-reactivities between HPV 6, 11, 16 and 45 were studied using polyclonal and monoclonal antibodies to virus-like particles, L1 proteins and synthetic peptides. Although antisera react strongly against homologous virus-like particles, there is evidence of some cross-reactivity. This could be one of the explanations for the fact that antibodies to one genotype are detected in individuals infected with another genotype. This study also identified a linear epitope recognized by anti-HPV 16 virus-like particle sera.

Cloning of human 2H9 heterogeneous nuclear ribonucleoproteins. Relation with splicing and early heat shock-induced splicing arrest.

Using antibody 2H9 from our heterogeneous nuclear ribonucleoproteins (anti-hnRNP) monoclonal antibody library, we previously showed in HeLa cells that a 35-37-kDa protein doublet switches from the hnRNP complexes to the nuclear matrix following a 10-min heat shock at 45 degrees C (1 Lutz, Y., Jacob, M., and Fuchs, J. P. (1988) Exp. Cell Res. 175, 109-124). cDNA cloning and sequencing revealed an hnRNP protein (2H9) which is a new member of the hnRNP F, H/H' family. Protein 2H9 displays two consensus sequence-type RNA binding domains (CS-RBD) showing 80-90% homology with two of the three CS-RBDs of hnRNP F and H/H'. Another common feature is the presence of two glycine/tyrosine-rich auxiliary domains located at the C terminus and between the two CS-RBDs. At the functional level we show that specific anti-2H9 peptide antibodies can directly inhibit an in vitro splicing system. Moreover, the 2H9 protein doublet is no more present in nuclear extracts from such briefly stressed cells, which interestingly correlates with the inability of these extracts to catalyze in vitro splicing reactions. Taken together, our data suggest that these proteins are involved in the splicing process and also participate in early heat shock-induced splicing arrest by transiently leaving the hnRNP complexes. These 2H9 proteins, which are encoded by a single gene located on human chromosome 10, were also found to be associated with nuclear bodies in situ.

The human hnRNP-M proteins: structure and relation with early heat shock-induced splicing arrest and chromosome mapping.

With anti-hnRNP monoclonal antibody 6D12 we previously showed in HeLa cells that as early as 10 min after the onset of a heat shock at 45 degrees C, a 72.5-74 kDa antigen doublet leaves the hnRNPs and strongly associates with the nuclear matrix, the effect being reversed after a 6 h recovery at 37 degrees C. cDNA cloning and sequencing enabled us to identify these antigens as hnRNP-M proteins and further to show that the correct sequence differs by an 11 amino acid stretch from the originally published sequence. We also show that monoclonal antibodies raised against synthetic hnRNP-M peptides can directly inhibit in vitro splicing. Furthermore, stressing cells at 45 degrees C for 10 min is sufficient to abolish the splicing capacity of subsequently prepared nuclear extracts which, interestingly, do not contain the hnRNP-M proteins any more. Taken together, our data suggest that these proteins are involved in splicing as well as in early stress-induced splicing arrest. Further in situ hybridization assays located the hnRNP-M encoding gene on human chromosome 19.

Two hnRNP-associated proteins share common structural features with the adenoviral 72-kDa protein.

Using our anti-hnRNP monoclonal antibody library Y. Lutz, M. Jacob, and J.-P. Fuchs (1988) Exp. Cell Res., 175, 108-124; P. Mähl, Y. Lutz, E. Puvion, and J.-P. Fuchs, (1989) J. Cell Biol. 109, 1921-1935, we investigated by immunocytofluorescence the fate of a series of speckled-distributed nuclear antigens, after HeLa cells were infected with adenovirus type 2. Although the speckled pattern, which corresponds to the nucleoplasmic fibrillogranular network, including the interchromatin-granule clusters, was still observed during most of the infectious cycle, several antibodies also revealed additional, increasingly fluorescent virus-induced structures. In noninfected cells, two of these antibodies, termed 3F2 and 2A5, recognize two antigens of 33 and 31 kDa, respectively. Western blot analysis showed that this increasing amount of fluorescence observed in infected cells did not reflect an accumulation of the 33- and 31-kDa antigens, but is actually due to the fact that both antibodies also recognize the multifunctional adenovirus 72-kDa single-stranded DNA-binding protein (DBP). Immunoelectron microscopy analyses, including sequential double-labeling, indeed showed that this additional signal precisely colocalizes with the viral 72-kDa DBP, which essentially accumulates over the entire surface of the virus-induced single-stranded DNA accumulation sites. Taken together, our data show that two host-specific hnRNP-associated antigens share common epitopes with the viral 72-kDa DBP.