Priming with secreted glycoprotein G of respiratory syncytial virus (RSV) augments interleukin-5 production and tissue eosinophilia after RSV challenge

The respiratory syncytial virus (RSV) G glycoprotein promotes differentiation of type 2 CD4+ T lymphocytes and induces an eosinophilic response in lungs of RSV-infected mice. A unique feature of G is that a second initiation codon in the transmembrane region of the glycoprotein results in secretion of soluble protein from infected cells. Recombinant vaccinia viruses that express wild-type G (vvWT G), only secreted G (vvM48), or only membrane-anchored G (vvM48I) were used to define the influence of G priming on immunopathogenesis. Mice immunized with vvM48 had more severe illness following RSV challenge than did mice primed with vvWT G or vvM48I. Coadministration of purified G during priming with the construct expressing membrane-anchored G shifted immune responses following RSV challenge to a more Th2-like response. This was characterized by increased interleukin-5 in lung supernatants and an increase in G-specific immunoglobulin G1 antibodies. Eosinophils were present in the infiltrate of all mice primed with G-containing vectors but were greatest in mice primed with regimens including secreted G. These data suggest the form of G protein available for initial antigen processing and presentation is an important factor in promoting Th2-like immune responses, including the induction of lung eosinophilia. The ability of RSV to secrete G protein may therefore represent a viral strategy for immunomodulation and be a key determinant of disease pathogenesis.     

A model for respiratory syncytial virus (RSV) infection based on experimental aerosol exposure with bovine RSV in calves

Five conventionally kept calves aged between 17 and 24 days were experimentally infected with bovine respiratory syncytial virus (BRSV) by aerosol in order to mimic the natural infection route. The calves were killed and autopsies performed 7 days after the first virus challenge. The BRSV isolate used induced tracheitis, bronchitis and atelectasis in infected calves. The only virus which could be isolated from the lungs of the calves was BRSV. In addition, Mycoplasma bovirhinis was isolated from the lungs or/and trachea of two calves. The clinical and histopathological findings, as well as the detection of BRSV antigens by immunofluorescence in the epithelial cells of lung and trachea, and the reisolation of the virus from bronchoalveolar lavage fluids of all inoculated calves, provided confirmation of successful infection with BRSV.     

Recombinant human respiratory syncytial virus (RSV) from cDNA and construction of subgroup A and B chimeric RSV

OBJECTIVE: The present study was performed to discriminate between central and peripheral effects of noradrenaline (NA) in normotensive, non-obese, type 2 diabetic patients. METHODS: Study I: In 10 patients and 10 healthy volunteer (HV) cumulative doses of NA were infused intravenously until mean arterial pressure (MAP) rose with 20 mmHg, and subsequently the effects on the forearm blood flow (FBF) was measured. Also, the FBF response to intra-arterial NA (0.025, 0.1, 0.4 micrograms min-1) was measured. Study II: In 13 patients and 14 HV the venous constrictor response to a cumulative local infusion of NA in a dorsal hand vein was determined. RESULTS: In study I the circulating plasma NA concentrations inducing a rise in MAP of 20 mmHg, were lower in the type 2 patients relative to the HV (p < 0.01). The relationship between changes in pressure and changes in heart rate were similar in both groups. Moreover, FBF responsiveness to intra-arterial NA was not different between the two groups. The slopes of the delta MAP/NA regression lines were correlated with basal insulin levels and relative insulin resistance in the healthy volunteers (R = 0.77, p < 0.01, and R = 0.83, p < 0.01), but not in the type 2 diabetic patients. In study II no differences were observed in the dose generating half maximum (ED50) and the maximum (Emax) response to NA between the type 2 patients and the HV. CONCLUSIONS: Non-obese normotensive type 2 patients have an increased pressor response to NA, which is not based upon a defect in skeletal muscle resistance arterioles, peripheral veins, or a defect in the baroreceptor system. Therefore, in type 2 diabetes the noradrenergic responsiveness of other vascular beds, such as the splanchnic or renal, must be enhanced.     

Detection of respiratory syncytial virus (RSV) antigen in the lungs of guinea pigs 6 weeks after experimental infection and despite of the production of neutralizing antibodies

Infections with respiratory syncytial virus (RSV) are characterized by frequently occurring reinfections and are regarded to be responsible for bronchial hyperreactivity. In this report we describe a small-animal model suited to study RSV-induced pathogenesis and immune response. Guinea pigs are infected by inhalation of an RSV-aerosol. Lungs of infected animals show signs of a bronchiolitis at 7 days after the initial infection. Although neutralizing serum antibodies are synthesized viral proteins are still detectable at 6 weeks post infection. Therefore, the presence of neutralizing antibodies is obviously not sufficient for rapid clearance of persistent RSV-proteins from the lungs of infected guinea pigs.     

Recombinant respiratory syncytial virus (RSV) bearing a set of mutations from cold-passaged RSV is attenuated in chimpanzees

A set of five missense mutations previously identified by nucleotide sequence analysis of subgroup A cold-passaged (cp) respiratory syncytial virus (RSV) has been introduced into a recombinant wild-type strain of RSV. This recombinant virus, designated rA2cp, appears to replicate less efficiently in the upper and lower respiratory tracts of seronegative chimpanzees than either biologically derived or recombinant wild-type RSV. Infection with rA2cp also resulted in significantly less rhinorrhea and cough than infection with wild-type RSV. These findings confirm the role of the cp mutations in attenuation of RSV and identify their usefulness for inclusion in future live attenuated recombinant RSV vaccine candidates.     

Immunological properties of plaque purified strains of live attenuated respiratory syncytial virus (RSV) for human vaccine

Respiratory syncytial virus (RSV) causes severe lower respiratory tract disease in infants, young children, and the elderly. Efforts to develop satisfactory live or inactivated vaccines have not yet been proven successful. Our research focuses on the development of four purified live attenuated RSV sub-type A human vaccine clones. Temperature sensitive (ts) and attenuated purified clones of either cold-adapted (ca) RSV or high-passage (hp) RSV were administered intra-nasally (i.n.) to BALB/c mice and tested for immunogenicity. All four clones produced significant anti-RSV F IgG2a and IgG1 titres in the sera of mice, RSV-specific neutralizing titres higher than those produced by their wild-type progenitor viruses, cytotoxic T-lymphocyte (CTL) activity, and total protection against wild-type (wt) viral challenge. These purified vaccine candidates await testing in humans to determine which contain the required balance between immunogenicity and attenuation.     

Production of env-deficient rous sarcoma virus (RSV) early after infection

In this paper we document a case of peripheral T cell lymphoma (PTCL) that exhibited variable T cell histology at presentation and follow-up. Southern blot analysis for T cell receptor (TCR) and immunoglobulin (Ig) receptor gene rearrangements failed to reveal clonal T or B cell populations. TCR gamma (TCRG) and beta (TCRB) chain gene polymerase chain reaction (PCR) amplification of DNA isolated from biopsies was also consistent with polyclonal T cell populations, however Ig PCR revealed clonal Ig rearrangements in follow-up biopsies but not in the presentation biopsy. There was no histological evidence for a neoplastic B cell population in these biopsies although occasional EB virus positive blasts were present. The significance of a cryptic B cell clone is unknown but suggests a relationship with the proliferating polyclonal T cells in this case of PTCL. These data reflect the complexity of PTCL with implications for treatment and patient management.     

CD8+ T cells control Th2-driven pathology during pulmonary respiratory syncytial virus infection

BALB/c mice vaccinated with vaccinia virus expressing the major surface glycoprotein G of respiratory syncytial virus (RSV) develop lung eosinophilia during RSV challenge. The G protein is remarkable in that it induces CD4+, but no CD8+ T cells in this mouse strain. Studies using passive T cell transfers show that co-injection of CD8+ T cells greatly reduces the Th2-driven lung eosinophilia caused by G-specific CD4+ T cells. By contrast, vaccination with the fusion protein (F) induces both CD8+ and CD4+ T cells, but not lung eosinophilia during RSV infection. These observations suggest that CD8+ T cells play a crucial role in preventing Th2-driven pathology. We therefore depleted mice with anti-CD8 antibodies in vivo. This treatment allowed lung eosinophilia to develop in F-primed mice. Depletion of interferon (IFN)-gamma had a similar effect, suggesting that secretion of this cytokine is the mechanism by which CD8+ T cells exert their effect. To test whether similar effects occurred in other strains of mice, RSV-infected C57BL/6 mice (which do not develop eosinophilia after sensitization to G) were treated with anti-IFN-gamma. Again, these mice developed eosinophilia. In this strain, genetic deletion of CD8-alpha, beta2-microglobulin or genes coding for the transporter associated with antigen presentation (which in each case eliminates CD8+ T cells) caused lung eosinophilia during RSV infection. These studies show the critical roles that CD8+ T cells and IFN-gamma production play in regulating Th2-driven eosinophilia and provide a unifying explanation for previous studies of lung eosinophilia. We propose that vaccines designed to enhance CD8+ T cell recognition might avoid disease caused by CD4+ Th2 cells.     

Immune response to baculovirus expressed protein fragment amino acids 190-289 of respiratory syncytial virus (RSV) fusion protein

At least two neutralizing epitopes have been identified in the amino acid (aa) sequence 190-289 of the RSV fusion protein. The authors expressed this region in insect cells (bF190-289) and compared the immune response to bF190-289 with that induced by baculovirus expressed full-length fusion protein (bF). As with bF, mice primed with bF190-289 produced exclusively antibodies of IgG1 isotype, generated neutralizing antibodies, reduced significantly the virus titer (about a half log10 reduction) after RSV challenge and induced a Helper T (Th) 2 cell response in mediastinal lymph node cells (MLNC) restimulated in vitro. Thus, the aa sequence 190-289 represents a major immunogenic region of the RSV fusion protein.     

Respiratory syncytial virus infection of human respiratory epithelial cells up-regulates class I MHC expression through the induction of IFN-beta and IL-1 alpha

CD8+ T cells mediate some of the damage to the lung epithelium following respiratory syncytial virus (RSV) infection. Since CD8+ T cells recognize antigen-laden class I MHC molecules on the target cells, we examined in this study the expression of class I MHC by RSV-infected respiratory epithelial cells. Respiratory epithelial cell lines and bronchial epithelial cells from normal human tissue responded to RSV infection with an increased expression of class I MHC as determined by flow cytometry and immunoprecipitation of class I MHC from metabolically radiolabeled cells. The increase in class I MHC expression was dependent on infectious, replicating virus. UV-irradiated culture supernatants from RSV-infected A549 cells, when added to fresh A549 cell cultures, induced an increase in class I MHC expression by those cells. The class I MHC increasing activity within supernatants from A549 cells was due, in large part, to IFN-beta, and to a lesser extent to IL-1 alpha. The addition of neutralizing Abs to both cytokines completely blocked the increase in class I MHC expression by cells treated with the above-mentioned supernatants. These results demonstrate that RSV infection elicits IFN-beta production by respiratory epithelial cells, which in turn leads to an increase in their synthesis of class I MHC, which would facilitate their recognition and lysis by RSV-specific CD8+ T cells.     

Detection and quantitation of human respiratory syncytial virus (RSV) using minigenome cDNA and a Sindbis virus replicon: a prototype assay for negative-strand RNA viruses

African trypanosomes undergo antigenic variation of their variant surface glycoprotein (VSG) coat to avoid being killed by their mammalian hosts. The active VSG gene is located in one of many telomeric expression sites. Replacement of the VSG gene in the active site or switching between expression sites can give rise to a new VSG coat. To study Trypanosoma brucei VSG expression site inactivation rather than VSG gene switching, it is useful to have an in vitro negative-selection system independent of the VSG. We have achieved this aim by using a viral thymidine kinase (TK) gene. Following integration of the TK gene downstream of the 221a VSG expression site promoter, transformant cell lines became sensitive to the nucleoside analog 1-(2-deoxy-2-fluoro-8-D-arabinofuranosyl)-5-iodouracil. These TK trypanosomes were able to revert to resistance at a rate approaching 10(-5) per cell per generation. The majority of revertants expressed a new VSG gene even though there had been no selection against the VSG itself. Analysis of these switched variants showed that some had shut down TK expression via an in situ expression site switch. However, most variants had the complete 221 expression site deleted and another VSG expression site activated. We speculate that a new VSG expression site cannot switch on without inactivation of the old site.     

Epidemiology of respiratory syncytial virus infection requiring hospitalization in East Denmark

BACKGROUND: Prophylaxis against infection caused by respiratory syncytial virus (RSV) with high titered RSV immunoglobulin or humanized antibody may soon be available in Europe. OBJECTIVE: To study the epidemiology of RSV infections requiring hospitalization in infants <6 months in East Denmark to provide a rational basis for decisions concerning prophylaxis against RSV. METHOD: Populat ion-based retrospective review of case records of infants <6 months admitted to pediatric departments with RSV infection in East Denmark from November 1, 1995, to April 30, 1996. RESULTS: Data were obtained from 459 infants. Seventy-three had predisposing conditions: prematurity, 49; pulmonary disease, 2; congenital heart disease, 7; neurologic disease, 6; others, 9. One preterm infant had bronchopulmonary dysplasia. The incidence of RSV infection requiring hospitalization in East Denmark among infants <6 months was estimated to be 34/1000/season. It was 32/1000/season among term infants and 66/ 1000/season among preterm infants (P<0.001). Infants with predisposing conditions and/or nosocomial infection (n = 24) had significantly more severe courses than otherwise healthy infants (P<0.01). One-hundred thirty infants received respiratory support by nasal continuous positive airway pressure, but only six required mechanical ventilation. No infants died. CONCLUSION: The course of RSV disease in East Denmark was milder than reported elsewhere, possibly as a result of the low prevalence of bronchopulmonary dysplasia in Denmark. However, RSV constitutes a considerable burden to the Danish pediatric health care system, and therefore prophylaxis against RSV is desirable.     

Effect of compounds with antibacterial activities in human milk on respiratory syncytial virus and cytomegalovirus in vitro

In differential diagnosis of a delir also adverse effects of medicaments have to be taken into account beside other causes. We report a case of an agitated delir with nocturnal disturbance of consciousness, confusion, restlessness and sleeplessness. This delir existed exclusively during the therapy of a cutaneous herpes zoster with zovirax-pills which can only be explained by a causal connection--after exclusion of other causes. As a so far undescribed predisposition for neurotoxicity of oral therapy with acyclovir signs of vascular encephalopathy were found in the patient's cranial magnetic resonance imaging. The central nervous side effects of acyclovir were summarized shortly.     

Interleukin-18 (IFNgamma-inducing factor) induces IL-8 and IL-1beta via TNFalpha production from non-CD14+ human blood mononuclear cells

Over a 6-month period, 23 members of the International Microvascular Research Group participated in a prospective survey of their microvascular free-flap practice. Data were recorded with each case for 60 variables covering patient characteristics, surgical technique, pharmacologic treatment, and postoperative outcome. A total of 493 free flaps were reported with a representative demographic distribution for age, sex, indications for surgery, risk factors, flap type, surgical technique, and pharmacologic intervention. Mixed effects logistic regression modeling was used to determine predictors of flap failure and associated complications. The overall incidence of flap failure was 4.1 percent (20 of 493). Reconstruction of an irradiated recipient site and the use of a skin-grafted muscle flap were the only statistically significant predictors of flap failure, with increased odds of failure of 4.2 (p = 0.01) and 11.1 (p = 0.03), respectively. A postoperative thrombosis requiring re-exploration surgery occurred in 9.9 percent of the flaps. The incidence of this complication was significantly higher when the flap was transferred to a chronic wound and when vein grafts were needed, with increased odds of failure of 2.9 (p = 0.02) and 2.5 (p = 0.02), respectively. There was a lower incidence of postoperative thrombosis when rectus/transverse rectus abdominis muscle (TRAM) flaps were used, where odds of failure decreased by 0.36 (p = 0.04), and when subcutaneous heparin was administered in the postoperative period, where odds decreased by 0.27 (p = 0.04). There was an overall 69-percent salvage rate for flaps identified with a postoperative thrombosis. Intraoperative thrombosis occurred in 41 cases (8.3 percent) and was observed more frequently in myocutaneous flaps or when vein grafts were needed (5.5 and 5.0 greater odds, respectively; p < 0.001) but was not associated with higher flap failure (2 of 41 cases; 4.9-percent failure rate). The incidence of a hematoma and/or hemorrhage was increased in obese patients and when vein grafts were needed [2.7 (p = 0.02) and 2.6 (p = 0.03) greater odds, respectively], whereas this complication was significantly decreased in muscle flaps (myocutaneous or skin-grafted muscle), in tobacco users, when a heparinized solution was used for general wound irrigation, and when the attending surgeon performed the arterial anastomosis (in contrast to the resident or fellow on staff) (p < 0.05 for each factor). With the multivariable analysis, many factors were found not to have a significant effect on flap outcome, including the recipient site (e.g., head/neck, breast, lower limb, etc.); indications for surgery (trauma, cancer, etc.); flap transfer in extremes of age, smokers, or diabetics; arterial anastomosis with an end-to-end versus end-to-side technique; irrigation of the vessel without or with heparin added to the irrigation solution; and a wide spectrum of antithrombotic drug therapies. These results present a current baseline for free-flap surgery to which future advances and improvements in technique and practice may be compared.     

An experimental study of a concurrent primary infection with bovine respiratory syncytial virus (BRSV) and bovine viral diarrhoea virus (BVDV) in calves

Experimental infections with bovine respiratory syncytial virus (BRSV) and bovine viral diarrhoea virus (BVDV) were performed to study the effect of concurrent BRSV and BVDV infections. Twelve seronegative calves, in 3 groups, were inoculated on a single occasion with pure BRSV (group A), BRSV and noncytopathogenic BVDV (group B) or mock infected (group C). Mild respiratory symptoms were recorded 4 to 5 days post inoculation (dpi) in group A and group B calves. One calf in group A was severely affected and required medical treatment. In group B, fever (40.7-41.4 degrees C) was prominent 7 to 8 dpi. Only calves in group B were BVDV positive in purified lymphocytes at 2 to 14 dpi and showed increased serum interferon levels, with a peak at 4 dpi, indicating BVDV to be responsible for inducing the rise. BRSV was detected in lung lavage fluids up to 7 dpi for group A calves, compared to 11 dpi for group B and calves in this group also seroconverted later displaying lower BRSV titers. The time lag before an antibody response and the titers recorded in group B, indicated that the duration of BVDV infection in lymphocytes negatively influenced the capacity to mount a BRSV antibody response.     

Lactobacilli and streptococci induce interleukin-12 (IL-12), IL-18, and gamma interferon production in human peripheral blood mononuclear cells

Human peripheral blood mononuclear cells (PBMC) were stimulated with three nonpathogenic Lactobacillus strains and with one pathogenic Streptococcus pyogenes strain, and cytokine gene expression and protein production were analyzed. All bacteria strongly induced interleukin-1beta (IL-1beta), IL-6, and tumor necrosis factor alpha mRNA expression and protein production. S. pyogenes was the most potent inducer of secretion of IL-12 and gamma interferon (IFN-gamma), and two of three Lactobacillus strains induced IL-12 and IFN-gamma production. All strains induced IL-18 protein production. IL-10 and IL-4 production was induced weakly and not at all, respectively. Our data show that nonpathogenic lactobacilli and pathogenic streptococci can induce Th1 type cytokines IL-12, IL-18, and IFN-gamma in human PBMC.     

Increased immune activation precedes the inflection point of CD4 T cells and the increased serum virus load in human immunodeficiency virus infection

In sickness-conditioned learning, animals become ill after sampling a new substance and develop an aversion that is expressed as avoidance of that substance in subsequent presentations. We examined the parameters of a one-trial, nongustatory, sickness-conditioned learning task in day-old chicks. Chicks pecked a bead and were made ill by i.p. injection of lithium chloride (LiCl). Both 0.5 and 1.0 M LiCl (0.1 ml) produced reliable avoidance at test. Chicks injected with LiCl between 15 and 45 min after training avoided the bead at test, whereas those injected within 5 or 10 min or more than 45 min after training did not. Avoidance was present until 24 h posttraining and absent after 48 h. Therefore, robust learning of the sickness-conditioned learning task occurs in one trial without the need for gustatory cues, and memory for the task lasts at least 24 h. Uses of this task to study memory formation in the day-old chick are discussed.