Interaction cloning and characterization of RoBPI, a novel protein binding to human Ro ribonucleoproteins.

Human Ro ribonucleoproteins (RNPs) are autoantigenic particles of unknown function(s) that consist of a 60-kDa protein (Ro60) associated with one hY RNA (hY1-5). Using a modified yeast three-hybrid system, named RNP interaction trap assay (RITA), we cloned a novel Ro RNP-binding protein (RoBPI), based on its property to interact in vivo in yeast with an RNP complex made of recombinant Ro60 (rRo60) protein and hY5 (rhY5) RNA. RoBPI cDNA contains three conserved RNA recognition motifs (RRM) and is present as a family of isoforms differing slightly at their 5' end. The 2.0-kb RoBPI mRNA was detected in all human tissues tested. Highly homologous cDNA sequences were found in banks of expressed sequence tags (ESTs) from mice. Two-hybrid, three-hybrid, and RITA experiments respectively established that 60 kDa RoBPI did not interact in yeast with rRo60 alone, with rhY5 RNA alone, or with bait RNPs consisting of rRo60 and recombinant hY1, hY3, or hY4 RNAs. RoBPI coimmunoprecipitated with Ro RNPs from HeLa cell extracts and partially colocalized with Ro60 in nuclei of cultured cells. Because hY5 RNA and RohY5 RNPs are recent evolutionary additions seen only in primates, but RoBPI seems more conserved, their interaction may represent a gain of function for Ro RNPs. Alternatively, interaction of RohY5 RNPs with RoBPI may have no functional bearing, but may underlie some of the unique biochemical and immunological properties of these RNPs.

Identification of ribonucleoprotein (RNP)-specific protein interactions using a yeast RNP interaction trap assay (RITA).

We describe an adaptation of the yeast three-hybrid system that allows the reconstitution in vivo of tripartite (protein-RNA-protein) ribonucleoproteins (RNPs). To build and try this system that we called RNP interaction trap assay (RITA), we used as a model the autoantigenic Ro RNPs. hY RNAs bear distinct binding sites for Ro60 and La proteins, and Ro RNPs are thus physiologically tripartite (Ro60/hY RNA/La). Using recombinant La (rLa) and Ro60 (rRo60) proteins and recombinant hY RNAs (rhY) co-expressed in yeast, we found that RNPs made of rRo60/rhY/rLa were readily reassembled. Reconstitution of tripartite RNPs was critically dependent on the presence of an appropriate Ro60 binding site on the recombinant RNA. The RITA assay was further used to detect (rRo60/rhY RNP)-binding proteins from a HeLa cell cDNA library, allowing specific identification of La and of a novel Ro RNP-binding protein (RoBPI) in more than 70% of positive clones. RITA assay may complement already available two- and three-hybrid systems to characterize RNP-binding proteins by allowing the in vivo identification of interactions strictly dependent upon the simultaneous presence of a protein and of its cognate RNA.

[Acquired pulmonary vein stenosis after surgery of complex partial anomalous pulmonary venous drainage in an adult. Diagnostic value of transesophageal echocardiography]. / Sténose acquise d'une veine pulmonaire après chirurgie d'un retour veineux pulmonaire anormal partiel complexe de l'adulte. Intérêt diagnostique de l'échocardiographie transoesophagienne.

Surgical correction of partial anomalous pulmonary venous drainage is difficult and may be complicated by acquired postoperative stenosis at the site of reimplantation of the pulmonary veins in the left atrium. Diagnosis should be made quickly because of the very poor prognosis due to acute pulmonary hypertension. The case described by the authors underlines the value of multiplane transesophageal echocardiography with two-dimensional and Doppler analysis for rapid and accurate diagnosis of this complication.

Analysis of T cell receptor beta chain expression by isoelectric focusing following gene amplification and in vitro translation.

We describe a new approach to analysis of T cell receptor diversity based on isoelectric focusing of in vitro translation products of amplified V region genes. The method is illustrated by analysis of V beta 2 profiles in peripheral blood lymphocytes from normal donors. The primers used for V beta 2 analysis spanned the V-(D-)J junction and included the segment from amino acid residue position 53 in the variable region to residue 132 of the constant region. The isoelectric focusing patterns display approximately 13-14 bands of varying intensity. Differences in expression of V beta 2-derived peptides were detected in comparisons of the isoelectric focusing profiles from different individuals, suggesting that the method may be useful for detecting genetically determined, immune response related or disease associated differences in Tcr V region expression. The major isoelectric focusing bands have been interpreted as representing groups of V beta 2 sequences sharing J beta region and NDN region charge similarity. Quantitative differences were detected in V beta 2 profiles of CD4 and CD8 T cell subpopulations indicating there may be selection for different charge characteristics in NDNJ sequences in the two T cell subsets. The method provides a new dimension for the detection of perturbations in the T cell repertoire.

[Antiphospholipid syndrome in children. Apropos of a case]. / Syndrome antiphospholipide chez l'enfant. A propos d'un cas.

The authors report the case of a 10 year old child who presented with an uncomplicated deep venous thrombosis associated with an antiphospholipid syndrome. The diagnosis was established by the finding of spontaneous prolongation of the activated cephalin time, the finding of a lupus-like antibody and an anti-cardiolipin antibody. The clinical outcome was good with oral anticoagulants but a recurrence was observed when they were stopped. The authors discuss the question of the duration of preventive therapy.

An in vitro assay for hepatitis C virus NS3 serine proteinase.

Hepatitis C virus (HCV) encodes a polyprotein of which the majority of nonstructural proteins are matured by the viral serine proteinase located in the N terminus of NS3. Intracellular studies using recombinant vaccinia virus have shown that both NS3 and its cofactor NS4A are required to enhance processing at the NS3-dependent cleavage sites. We developed an in vitro (cell-free) assay in which the HCV serine proteinase was shown to be enzymatically active, by mixing lysates of cells expressing either the serine proteinase or a nonstructural protein substrate. NS3 cleaved in a highly reproducible manner at the NS5A/5B site in the presence of NS4A, whereas NS3 alone was enzymatically inactive. NS4A could be provided either linked to NS3 or as part of the substrate. In contrast, irrespective of the presence or absence of NS4A, no NS3-mediated processing was observed at the NS3/4A, NS4A/4B, and NS4B/5A sites in this assay. In vitro cleavage at the NS5A/5B site occurred rapidly, within 1 min at temperatures ranging from 0 to 20 degrees, but was incomplete and required detergent-solubilized lysates. General serine proteinase inhibitors did not decrease processing activity. The in vitro model described in this study is a new tool: (1) to study the structure and the function of HCV serine proteinase and NS5A/5B cleavage site, and (2) to test NS3 serine proteinase inhibitors.

Infection of a leukemic cell line (K562) by hepatitis B virus induces cell growth inhibition.

Hepatitis B virus inhibits the in vitro growth of the human leukemic cell line K562; however, the mechanism of this growth inhibition is not understood. One to 12 days after exposure, viral DNA and RNA were detected in K562 cells by Southern blot and reverse-transcriptase polymerase chain reaction analyses. Virus-containing serum that was heat-inactivated failed to inhibit cell growth and no viral DNA or RNA was detected in these cells. In addition, murine monoclonal antibodies directed to hepatitis B virus surface epitopes neutralized the virus-induced growth inhibition whereas antibodies to hepatitis B virus core epitopes failed to suppress the inhibition. These results indicate that in vitro infection of K562 cells by hepatitis B virus causes inhibition of hematopoietic cell line growth.

Hepatitis C virus is detected in a monocyte/macrophage subpopulation of peripheral blood mononuclear cells of infected patients.

Hepatitis C virus (HCV) is the primary agent of posttransfusion non-A, non-B hepatitis. HCV RNA was detected in peripheral blood mononuclear cells (PBMC) by polymerase chain reaction in 17 of 24 HCV-infected patients with chronic hepatitis with or without cirrhosis. One of 5 patients whose PBMC contained HCV RNA also had negative-stranded HCV RNA in the PBMC. In 3 of 11 patients whose PBMC contained HCV RNA, flow cytometry with a murine monoclonal antibody to HCV core epitope revealed cytoplasmic staining of peripheral blood monocytes. The monocyte surface and the peripheral blood lymphocytes did not stain for HCV core epitopes. No correlation could be made between the presence of HCV RNA or antigen in PBMC and any serologic markers of HCV infection. These results indicate that monocyte uptake of HCV by either phagocytosis or infection may be part of the pathophysiology of this chronic disease.

Phytohemagglutinin and concanavalin A activate hepatitis B virus in peripheral blood mononuclear cells of patients with chronic hepatitis B virus infection.

Peripheral blood mononuclear cells (PBMC) from 25 patients with chronic hepatitis B were tested for the presence of free monomeric hepatitis B virus (HBV) DNA migrating as a single 3.2 Kb band by Southern blot analysis. The PBMC were cultured for 7 days in the presence of phytohemagglutinin (PHA) or concanavalin A (ConA) both of which yielded a proliferative response. By contrast, both bacterial lipopolysaccharide (LPS) and interleukin 2 (IL2) failed to do so. Dot blot assays were used to monitor HBV DNA level increase within PBMC. Following mitogen exposure HBV DNA levels increased above pre-stimulation levels in 19/25 PHA cultures, 6/15 ConA cultures, 1/15 LPS cultures, and 1/15 IL2 cultures. In 15 patients, Southern blot analysis was carried out before and after PHA exposure. In 13/15 cases, a single 3.2 Kb band was observed in unstimulated cultures as well as in PHA cultures even though PHA induced a HBV DNA increase. One case exhibited bands migrating faster than the 3.2 Kb signal, compatible with replicating intermediates and one case provided evidence of viral concatemers within PBMC after PHA stimulation. No HBV DNA was detected in the culture supernatants. The increase of HBV DNA level in PBMC induced by mitogen was strongly associated with an increase in HBV DNA expression (HBV RNA and HBs antigen). These studies indicate that HBV DNA present in human PBMC does represent a potential reservoir for infection with endogenous reactivation following PBMC activation.

Experimental studies and preliminary clinical trial of vinorelbine-loaded polymeric bioresorbable implants for the local treatment of solid tumors.

Vinorelbine is a new 5' nor Vinca alkaloid, active by i.v. route, in various types of cancer disease such as non-small cell lung cancer and advanced breast cancer. In order to evaluate the possibility of using this drug for local treatment of cancer, Vinorelbine-loaded bioresorbable polymeric implants were prepared using a copolymer of D,L-lactic and glycolic acids (PLA 37.5 GA 25). According to the manufacturing process, the 1.2-mm-diameter cylindrical rods obtained had a drug content of 1, 5, or 20% (w/w) and released half of their content within about 6 days in vitro. In vivo release in rats was slower, half of the drug being released after about 14 days. A dose-dependent antitumoral effect was observed in mice (solid P388 leukemia model) when implants were administered into or in contact with the tumor. At highest drug loads and when administered soon after tumor implantation, Vinorelbine implants were more effective than i.v. administration (median survival time of treated animals related to untreated controls, greater than 360 versus 188). In dogs, results of toxicity experiments revealed that administration of implants in vital organs must be avoided. On the contrary, s.c. administration was well tolerated. A transient local necrosis was observed in the days following implantation, but normal skin was recovered after about 10 weeks. Thus, a clinical trial was conducted on patients with head and neck cancer; implantation of 20% loaded polymeric implants into the tumor sites succeeded in 8 of 9 patients. The sole failure was attributed to the unusual hardness of the tumor tissue. Except for a local transient inflammatory reaction (easily treated with nonsteroidal antiinflammatory agents), no other sign of toxicity was detected, and patients tolerated the device well. Fourteen days after implantation, patients underwent their planned surgery, and the implants were recovered. Residual drug content varied from 24 to 55%. In all cases, there was a clearly delimited necrotic area around the implant, ranging from 0.5 to 3.5 cm in diameter. In the smallest tumors, necrosis was also observed in the normal tissue inside this area. These results invite further studies to evaluate such drug-loaded polymeric implants.

Detection of polyadenylated RNA in hepatitis B virus-infected peripheral blood mononuclear cells by polymerase chain reaction.

Polymerase chain reaction (PCR) with a reverse transcriptase step characterized a specific transcription activity in hepatitis B virus (HBV)-infected peripheral blood mononuclear cells (PBMC) in two patients (1 and 2) with chronic hepatitis positive for antibody to hepatitis B core antigen (HBc). Patient 1 was also coinfected with human hepatitis delta virus. A patient who cleared HBV replication after antiviral treatment with vidarabine served as negative control. HBV-specific RNA poly A sequences were detected by PCR in PBMC of patients 1 and 2 even without detectable HBV DNA (patient 2) as shown by dot blot and PCR assays. RNA sequences were found in both the nucleus and cytoplasm. The demonstration of HBV mRNA sequences within PBMC suggests the transcription of viral DNA, in agreement with the findings of HBV surface antigen in PBMC. The results in patient 1 demonstrated HBV mRNA sequences in leukocytes even without PCR-detectable HBV DNA sequences, likely due to ongoing hepatitis delta virus replication.

Detection of pre-S1 proteins in peripheral blood mononuclear cells from patients with HBV infection.

The presence of pre-S1 proteins in peripheral blood mononuclear cell (PBMC) samples from 115 patients with different forms of hepatitis B virus (HBV) infection was investigated by Western blot. Among 67 chronic HBsAg carriers, HBV antigens were detected in the PBMC in 80% for HBsAg, 27% for HBc/e Ag and 34% for pre-S1 proteins. The detection of pre-S1 proteins in PBMC was significantly associated with the presence of serum markers of HBV replication (HBV DNA and/or DNA polymerase). In the group of 48 consecutive patients negative for serum HBsAg, but positive for anti-HBc with or without anti-HBs, HBsAg and pre-S1 proteins could be detected in PBMC. This finding was more frequent among anti-HIV-positive patients (77 and 23% of the cases, respectively) than in the negative ones (23 and 4% of the cases, respectively). The detection of HBV DNA and polyadenylated RNA in some of the PBMC samples positive for HBV proteins suggests that these proteins may be expressed in PBMC, especially during intense HBV replication. In patients negative for serum HBsAg, PBMC may constitute a reservoir of HBV.

Different forms of hepatitis B virus DNA and expression of HBV antigens in peripheral blood mononuclear cells in chronic hepatitis B.

The presence of both hepatitis B virus (HBV) DNA and HBV antigens (HBsAg, HBeAg) was assessed in peripheral blood mononuclear cells (PBMC) from 32 patients chronically infected with HBV. Three different molecular forms of HBV DNA were observed: free monomers (5), high-molecular-weight free concatemers (11), and integrated HBV DNA (9). The HBV DNA patterns in the PBMC were different from those found in liver and did not correlate with any specific profile of serum HBV markers. When the same PBMC were assayed for HBsAg, 22 of the 25 HBV DNA positive samples, but only three of the seven HBV DNA negative samples, were positive. By contrast, none of the PBMC samples from five healthy HBV vaccine recipients gave any positive signal in the HBV DNA or HBsAg assays. In some patients, T and B cells, monocytes, and polymorphonuclear (PMN) cells were assayed separately, showing that the DNA pattern was similar for these different leucocytes subsets and ruling out the possibility that these patterns might reflect PMN cell contamination. Thus, in chronic HBV infection, 87.5% (28/32) of patients were found to contain at least one HBV marker in their PBMC, and a strong correlation was found between the presence of HBV DNA and viral antigens, suggesting a specific expression of HBV encoded proteins.

In vitro inhibition of hemopoietic cell line growth by hepatitis B virus.

The effects of hepatitis B virus (HBV) on established human cell lines of various tissue origins were evaluated by clonal or colorimetric assays in methylcellulose culture. HBV exposure inhibited the growth of six hemopoietic cell lines, while similar incubation did not affect the growth of seven nonhemopoietic carcinoma cell lines of breast, colon, liver, and stomach origin. The inhibition of hemopoietic cell line colony formation was dependent on the presence of intact viral (Dane) particles and the ratio of exposure of virions to cells and was reversible with antibodies to pre-S1, pre-S2, and S envelope protein epitopes. Purified HBV DNA, surface antigen pre-S antigens, and core antigen did not inhibit cell line growth. These results further demonstrate the tropism of HBV for cells of hemopoietic origin, confirming our previous findings on the effects of HBV on the growth of normal bone marrow progenitor cells in vitro. Established human tissue culture cell lines may be used to study the interactions of hemopoietic cells with HBV.

Prevalence and significance of hepatitis B virus antigens: expression in peripheral blood mononuclear cells in chronic active hepatitis.

One-hundred four chronic active hepatitis (CAH) patients were investigated for the expression of the hepatitis B virus (HBV) surface and core gene products (HBs Ag, HBc/HBe Ags) in peripheral blood mononuclear cells (PBMC). Two-thirds of 59 HBs antigenemic patients expressed HBs Ag in PBMC but 26% of cases positive for both anti-HBs and anti-HBc also expressed HBs Ag while none of the controls reacted. Among HBs antigenemic patients, only those who replicated HBV express the core gene products (HBc and/or HBe Ag) in PBMC, and high replicators did so more often than low replicators (P less than 0.05). The HBs Ag prevalence in PBMC, although slightly higher among HBe Ag/DNAp-positive cases could not be correlated with the intensity of HBV replication. In 16 cases (8 replicants and 8 nonreplicants) HBV DNA was detected by DNA hybridization spot test, while 8 controls devoid of HBV markers were negative. Both T and non-T cells reacted similarly for antigenic or genomic HBV markers. When the expression of HBV gene products in PBMC among 43 cases with HBs antigenemia was compared with that in the liver, a good correlation was found in 70% of cases for HBs Ag but in only 40% for HBc and HBe Ags. By contrast, among 38 cases lacking HBs Ag in the serum but positive for anti-HBc with or without anti-HBs, concordance between liver and PBMC expression of core gene products (69%) was better than for HBs antigenemic patients (40%). These data suggest that PBMC including T lymphocytes may represent the second-best HBV target and may mimic the steps of HBV cycle within hepatocytes.