Marta Zapata

Measles Virus-Specific Immunoglobulin G Isotype Immune Response in Early and Late Infections

ABSTRACT A total of 154 human serum samples (32 acute-phase and 22 convalescent-phase serum samples obtained within a week and between days 8 and 26 after the onset of rash, respectively, and 100 samples drawn from healthy immune adults) were processed by an immunofluorescence assay for the detection of immunoglobulin M (IgM), total immunoglobulin G (IgG), IgG1, IgG2, IgG3, and IgG4 measles virus-specific antibodies. In the acute phase, IgG1 was seen first, followed by IgG2, IgG3, and IgG4 responses, the mean seropositivity of which gradually increased during convalescence, reaching 100% (standard deviation [SD], 84 to 100%), 57% (SD, 34 to 80%), 86% (SD, 66 to 100%), and 86% (SD, 66 to 100%), respectively. IgG2 rose and fell in connection with IgG3 subclass antibodies, showing a rate of detection of IgG2 and/or IgG3 subclass antibodies of 95.5% (range, 100 to 86.5%) in the convalescent phase of infection. The mean percentage of measles IgG2 and IgG3 seropositivity dropped significantly during the memory phase, to 2% (range, 2 to 6%) and 3% (range, 3 to 7%), respectively (P < 0.05); meanwhile IgG1 and IgG4 subclass responses remained relatively unmodified in samples obtained years after infection (mean 100% [SD, 96 to 100%] and 86% [SD, 79 to 93%], respectively). Results obtained defined two highly different immune isotypic response patterns. One pattern is restrictive to IgG2 and/or IgG3 in the convalescent phase and is kinetically similar to the IgM antibody response, so its detection could be referred to as a recent viral activity. On the other hand, IgG1 and IgG4 were detected in both the convalescent and memory phases of the immune response, but their isolated occurrence without IgG2 and IgG3 could be related to the long-lasting immunity.

Rubella Virus Does Not Induce Apoptosis in Primary Human Embryo Fibroblast Cultures: A Possible Way of Viral Persistence in Congenital Infection

Congenital rubella is a persistent infection that contrasts with acute postnatal infection. Basis of the Rubella virus (RV) persistence still remain unknown, though several hypotheses have been postulated. RV induces apoptosis in cell lines, maybe as a way of cell-autonomous defense mechanism against virus. Considering the pattern of c-oncogenes expression during embryogenesis, which promotes proliferation while it inhibits apoptosis in specific cells, at certain times, it can be proposed that when RV infection establishes early in gestation, embryo cells that are proliferating have their apoptotic pathways shut down; then infected proliferating embryo cells cannot execute their apoptotic death program. We here report that RV induces apoptosis in human normal-term placenta chorionic villi explants (CVE) and in monolayers of cytotrophoblasts (CTB), but does not induce apoptosis in primary human embryo fibroblasts (HEF) cultures. These results suggest distinct responses to RV infection when comparing differentiated cells, as CTB, to cells with high proliferating potential, as HEF. RV shoots apoptosis in the former, whereas in fibroblastic dividing cells derived from embryo, RV appears not to be enough stimulus to activate the genetic program of cell death.

Isolation of Chlamydophila pneumoniae from atheromas of the carotid artery and their antibiotics susceptibility profile

BACKGROUND Atherosclerosis is pathogenically similar to a chronic inflammatory response. Peripheral arterial disease (PAD) is a common manifestation of atherosclerosis. Chlamydophila pneumoniae has been suggested to play a role in the origin of PAD. OBJECTIVE To determine whether C. pneumoniae is present in atherosclerosis lesions of the carotid artery wall in patients with PAD through several diagnostic methods and to characterize C. pneumoniae susceptibility profiles. METHODS The presence of C. pneumoniae in 9 tissue samples from atherosclerotic lesions obtained by carotid endarterectomy was investigated by 3 methods. Karnofsky-fixed specimens were examined by transmission electron microscopy (TEM), isolation of C. pneumoniae was attempted in LLCMK2 cell structure (ICC), and the presence of chlamydial DNA was investigated by polymerase chain reaction (PCR). The in vitro activities of azithromycin, roxithromycin and penicillin were tested in 4 isolations and the reference strain of C. pneumoniae (AR39). RESULTS C. pneumoniae was detected in atherosclerotic plaques from 4 patients with PAD. The pathogen was identified by TEM, PCR and ICC. We report data of the in vitro susceptibility of 4 strains. These strains did not differ from respiratory AR39 strain in their susceptibility patterns to azithromycin, roxithromycin and penicillin. CONCLUSIONS C. pneumoniae is frequently found in the advanced carotid atherosclerotic lesions of patients undergoing endarterectomy. Although these findings do not establish causality in carotid artery atherosclerosis, they should stimulate investigation of the possible causal or pathogenic role of C. pneumoniae. Notably, the profiles of antibiotic susceptibility of C. pneumoniae isolated from 4 of the patients did not differ from those of the reference strain.

Evaluation of Antibodies against a Rubella Virus Neutralizing Domain for Determination of Immune Status

ABSTRACT The protective immune responses against rubella virus (RV) are related to its neutralizing epitopes, an issue that is important to consider when assessing the immune status of patients with remote infection. In the present paper, we compare the antibodies detected by a synthetic-peptide-based enzyme immunoassay (EIA) with antibodies detected by the traditional technique of hemagglutination inhibition (HIA) in patients with remote RV infection. The synthetic peptide used as an antigen (SP15) represents a neutralizing epitope that corresponds to amino acids 208 to 239 of the E1 glycoprotein. The SP15-EIA was developed, all variables that affected the assay were standardized, and the test was validated using reference sera. Serum samples (n = 129) from patients with remote RV infection were tested by HIA and SP15-EIA. Discrepant sera were assayed by MEIA (IMX/Abbot). The comparison between HIA and SP15-EIA, taking HIA as the standard methodology for determining immune status, showed that SP15-EIA is very specific and sensitive for detecting protecting antibodies (specificity, 100%; sensitivity, 98.20%). This study demonstrates that antibodies against the neutralizing domain represented by SP15 would be important in the memory response after natural infection and may be a good tool in the determination of the true immune status of patients with remote infection with regard to RV.

Apoptosis induction by the infection with Gilchrist strain of rubella virus

Apoptosis is an active process of cellular self-destruction, which can be initiated in response to several stimuli such as toxic substances, hormones, cytokines, trophic or osmotic modifications and viral infections. In this study, we demonstrate that in vitro rubella-virus (RV) induced cell death exhibited properties of apoptosis, characterized by condensation and segmentation of nuclei and internucleosomal cleavage of nuclear DNA. Apoptosis was not seen in the cells absorbed with UV-inactivated virus, indicating that the viral replication is required for the induction of apoptosis. Our results suggest that most of the cells undergoing apoptosis are non-infected neighboring cells.

Cell-fusion assay for the detection of rubella virus in Vero cells

BACKGROUND AND OBJECTIVES Rubella virus (RV) produces a subtle and slow-developing cytopathic effect in Vero cells that is difficult to recognize, especially at low multiplicities of infection. In order to facilitate the detection of RV in cell culture, we standardized a low-pH virus-mediated cell-fusion assay. STUDY DESIGN The incubation periods, temperatures, pH and multiplicity of infection were established. The specificity of the method was tested by immunofluorescence assay and cell-fusion inhibition by specific sera. RESULTS Six days post infection, Vero cells were treated for 5 min with fusion medium. After that, monolayers were incubated with medium at neutral pH for 16 h and then stained. Gigantic cells with multiple nuclei were observed. CONCLUSIONS The method allowed the observation of unequivocal images that are easier to recognize than the cytopathic effect caused by RV in the same cell line. At the same time, the method is simple, accessible and shown to be specific to demonstrate the replication of several strains and isolates of RV in Vero cells.

Analysis of viral glycoproteins by glycosidic digestion inside a polyacrylamide gel.

We adapted the method described by Cleveland et al. (1977); (Peptide mapping by limited proteolysis in sodium dodecyl sulphate and analysis by gel electrophoresis. J. Biol. Chem. 252, 1102-1106) to study the glycosidic residues linked to the viral glycoproteins of two enveloped viruses: Junin virus (JV) and rubella virus (RV). Radioiodinated glycoproteins were obtained from purified virions, isolated from SDS-polyacrylamide gels and then hydrolysed by specific glycosidases inside a second gel. N-linked oligosaccharides, mannose and galactose were found as terminal residues in the JV-G1 glycoprotein. Mannose and N-glycans of complex hybrid type were present on RV glycoproteins.

Kinetics of rubella-specific IgM antibody response in postnatal rubella infection

The serological diagnosis of primary postnatal rubella infection is based on detection of rubella-virus-specific IgM antibody or a four-fold rise in rubella-specific IgG antibody. Although there are several different methods of enzyme immunoassays that are commercially available, the cost benefit evaluation makes them impractical for use in developing countries. For this reason, we have standardized the measurement of rubella IgM antibody by HAI following serum fractionation by ion-exchange chromatography. The sera samples obtained from pregnant women infected with rubella virus at different times during gestation were fractionated and tested by HAI. Seven out of nine sera collected within the first two days after onset of rash showed detectable levels of rubella IgM antibody. All 57 sera collected between 3 and 30 days after the onset of rash contained rubella IgM antibody. After 30 days, only 1 of 5, or 20%, of sera contained IgM antibody. The HAI testing method was rapid and specific and the cost was not prohibitive. HAI-IgM testing could be used to diagnose primary rubella infections in developing countries where expensive EIAs are unaffordable.

Mouse brain gangliosides. Their property of inhibiting EEE virus hemagglutination

Gangliosides isolated from the brain of ten-day-old Swiss albino mice were purified by thin layer chromatography, and the resulting purified gangliosides were tested for their ability to inhibit hemagglutination by Eastern Equine Encephalitis virus. The trisialoganglioside and the more rapidly migrating of the two disialogangliosides were found to inhibit hemagglutination. It is postulated that the neuraminic acid moiety of these gangliosides is necessary for the hemagglutination inhibition property.