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Maternal expression of the checkpoint protein BubR1 is required for synchrony of syncytial nuclear divisions and polar body arrest in Drosophila melanogaster.

The spindle checkpoint is a surveillance mechanism that regulates the metaphase-anaphase transition during somatic cell division through inhibition of the APC/C ensuring proper chromosome segregation. We show that the conserved spindle checkpoint protein BubR1 is required during early embryonic development. BubR1 is maternally provided and localises to kinetochores from prophase to metaphase during syncytial divisions similarly to somatic cells. To determine BubR1 function during embryogenesis, we generated a new hypomorphic semi-viable female sterile allele. Mutant females lay eggs containing undetectable levels of BubR1 show early developmental arrest, abnormal syncytial nuclear divisions, defects in chromosome congression, premature sister chromatids separation, irregular chromosome distribution and asynchronous divisions. Nuclei in BubR1 mutant embryos do not arrest in response to spindle damage suggesting that BubR1 performs a checkpoint function during syncytial divisions. Furthermore, we find that in wild-type embryos BubR1 localises to the kinetochores of condensed polar body chromosomes. This localisation is functional because in mutant embryos, polar body chromatin undergoes cycles of condensation-decondensation with additional rounds of DNA replication. Our results suggest that BubR1 is required for normal synchrony and progression of syncytial nuclei through mitosis and to maintain the mitotic arrest of the polar body chromosomes after completion of meiosis.

Alleles↗

Apoptosis cascade progresses during turnover of human trophoblast: analysis of villous cytotrophoblast and syncytial fragments in vitro.

Growth and survival of the trophoblast layer of the human placenta depends on continuous incorporation of villous trophoblast stem cells (cytotrophoblast), by syncytial fusion, into the syncytiotrophoblast. Descriptive studies suggest that this process may be intimately related to apoptosis. We have analyzed the expression and activation of initiator and execution caspases, critical effectors of apoptosis, in relation to trophoblast turnover (differentiation) in human placental villi. We used immunohistochemistry, caspase enzyme histochemistry, caspase activity assays, Western blots, and autoradiography techniques on placental tissue sections, trophoblast lysates, villous explants, and isolated trophoblast fragments (villous cytotrophoblast and mononuclear syncytial elements) in vitro. Our data demonstrate expression of initiator caspases 8 and 10 and activity of caspase 8 in villous cytotrophoblast. Proforms of the execution caspases 3, 6, and 7 were also expressed in villous cytotrophoblast, but activation of execution caspases 3 and 6 could only be demonstrated in the syncytiotrophoblast after syncytial fusion. Down-regulation of the general transcription level (reduced incorporation of [3H]uridine) as well as cleavage of the execution caspase substrates poly-(ADP-ribose)polymerase and lamin B was confined to syncytiotrophoblast and preceded the final events of apoptotic death (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling reactivity and nuclear collapse). Our data confirm that the apoptosis cascade in villous trophoblast is regulated in parallel with trophoblast differentiation, syncytial fusion, and trophoblast turnover.

Apoptosis↗

Syncytial units isolated from early human placentas.

This morphological study demonstrates that during the early phases of pregnancy, in the syncytium there are individual multinucleated units joined to one another by junctional complexes which probably are destined to flow together with the main syncytium by membrane breakdown. Normal human placentas from 6th to 12th week of pregnancy, derived from voluntary abortions, have been utilized. According to the method described by Tedde et al. (1991), clusters of villi have been isolated, washed in saline and subjected to a treatment with a 0.3% trypsin solution that caused the detachment of numerous cellular elements from the surface of the free villi. Such isolated elements have been subjected to centrifugation and separation in Percoll gradients. Then multinucleated syncytial units have been collected, attached to slides by cytocentrifugation, stained with toluidine blue, observed at the light microscope and utilized to count their nuclei: about 600 elements from different weeks of pregnancy have been considered and the results have been submitted to statistical analysis. The svncytial units are multinucleated and can often be different from one another, particularly in regard to the characteristics and the number of the nuclei. The evaluation of the number of nuclei contained in different syncytial elements shows that nuclei are less numerous in the early phases (6-8 weeks) and increase in number towards the end of the first trimester in a statistic significant way. In conclusion, the present study could confirm the existence of single syncytial units, joined to constitute the syncytial layer, probably destined to a complete fusion in the main syncytium and likely in functional relationship with the underlying Langhans cells.

Cell Nucleus↗

Formation of vasculo-syncytial membranes in the human placenta.

Vasculo-syncytial membranes are localised areas of the placental villous membrane where the thickness of the barrier separating the maternal and fetal circulations is reduced to as little as 1-2 microns. Consequently, they are believed to be important sites for diffusional exchange. The morphological appearances suggest that they are caused by the obtrusion of locally dilated segments of the fetal capillaries into the trophoblast layer. This study sought quantitative evidence for the hypothesis by performing stereological analyses on vasculo-syncytial membranes at the electron microscopic level. The results confirmed that a strong relationship existed between the thickness of the capillary endothelium and that of the overlying stromal and trophoblastic tissue at these sites (r = 0.47, P < 0.001), indicating that some asymmetrical stretching or remodelling of the capillary wall was involved. Comparisons were also made between the thickness of the trophoblastic, stromal and endothelial components of the villous membrane in villi obtained from the central and from the peripheral parts of placental lobules, where vasculo-syncytial membrane formation is accentuated. The mean thickness of each component was lowest in the samples from the peripheral region, although the differences only proved to be statistically significant for the stromal layer (P = 0.01). Both sets of data lend quantitative support to the hypothesis that vasculo-syncytial membrane formation is the result of obtrusion of locally dilated segments of the fetal capillaries. The way in which this may be linked to changes in the dynamics of the fetal circulation as gestation advances is discussed.

Capillaries↗

Syncytial knots and intervillous bridges in the human placenta: an ultrastructural study.

An electron microscopic study has shown that the syncytial knots of the villi of the human placenta contain aggregated nuclei which exhibit marked degenerative changes; within the cytoplasm there is an abundance of cytoplasmic filaments and many stacks of annulate lamellae. It is suggested that syncytial knots are a sequestration phenomenon in which senescent nuclear material is aggregated and removed from metabolically active areas of the syncytiotrophoblast. Intervillous bridges appear to be formed chiefly by fusion of syncytial knots from adjacent villi, and it seems reasonable that the effete material in a syncytial knot should be used for this purpose. The intervillous bridges hava a fine structure which suggests that they have a mechanical function, and this lends support to the theory that they form an internal strut system within the placenta.

Cell Aggregation↗

Antigenic diversity of respiratory syncytial viruses and its implication for immunoprophylaxis in ruminants.

Bovine respiratory syncytial virus (BRSV) is a very important pathogen of cattle and perhaps other ruminants. It is a major contributor to the incidence of respiratory tract disease in nursing beef and feedlot and dairy calves. The genome of respiratory syncytial viruses encodes 10 proteins translated from 10 unique mRNAs. The major glycoprotein (G), fusion protein (F), 1A protein and the 22K protein are components of the viral envelope. The nucleocapsid contains the nucleocapsid protein (N), the phosphoprotein (P), and the large protein (L). The matrix protein (M) forms a structural layer between the envelope and the nucleocapsid. Antibodies to all the structural proteins develop in convalescent calves. However, evidence suggests that immunity develops primarily as a result of the antigenic stimulus by the major glycoprotein G and the fusion glycoprotein F. It is known also that activated cytotoxic T cells interact with N and F protein antigens and helper T cells interact with N, F, and 1A protein antigens. With the exception of the major glycoprotein, the respective proteins of various respiratory syncytial viruses share major antigenic domains. Based on antigenic differences of the major glycoprotein, at least 3 subgroups of RSV are recognized; human A, human B, and bovine RSV. Indirect evidence suggests that a second subgroup of BRSV exists. However, we have identified only one BRSV subgroup based on our work with RNase mismatch cleavage analysis of the G protein gene from a limited number of strains. Furthermore, our data indicated that a caprine RSV isolate is closely related to the bovine strains, but an ovine isolate is not. The latter may constitute yet another subgroup of RSV. These data affect decisions on optimization of immunoprophylaxis since evidence suggests that protection against a homologous RSV subgroup virus is superior to that against a heterologous strain in immune subjects.

Animals↗

[Acute respiratory tract infections due to a human metapneumovirus in children: descriptive study and comparison with respiratory syncytial virus infections].

BACKGROUND: A new paramyxovirus, the human metapneumovirus was recently isolated. We report the first French cases collected between 2000 and 2002. MATERIAL AND METHODS: Samples were obtained from nasopharyngeal aspirates from children hospitalised for acute respiratory tract infection in hospitals of Caen and Flers in Basse-Normandie. Human metapneumovirus was studied by polymerase chain reaction on negative samples for respiratory syncytial virus, influenza A and B virus, parainfluenza (1, 2 and 3) virus, adenovirus, coronavirus and rhinovirus. Comparison between metapneumovirus virus and respiratory syncytial virus infections was done after matching sex, age and infection month. RESULTS: Twenty-six human metapneumovirus infections were identified. A comparative study of a matched group of children infected by respiratory syncytial virus found no significative difference for hospitalisation motive, clinical criteria and treatment. CONCLUSION: The human metapneumovirus is responsible for typical acute bronchiolitis in children.

Acute Disease↗

A comparison of Binax NOW to viral culture and direct fluorescent assay testing for respiratory syncytial virus.

The Binax NOW immunochromatographic assay for respiratory syncytial virus was prospectively compared with direct fluorescent assay and viral culture at Primary Children's Medical Center, Salt Lake City, Utah during February 2003. Three hundred ten patient specimens were collected for testing, of which 102 specimens were positive for respiratory syncytial virus by the reference tests, direct immunofluorescence assay (DFA), and culture or molecular analysis. DFA analysis identified an additional 40 patient specimens positive for other respiratory viruses. Compared to the reference tests, the sensitivity, specificity, and positive and negative predictive values of the rapid immunochromatographic assay for detection of respiratory syncytial virus were 89.2%, 100.0%, 100.0%, and 94.9%, respectively. This rapid assay format proved to be cost-effective and simple to use in comparison to DFA and viral culture. Negative rapid test results should still be confirmed with a secondary test.

Adolescent↗

Effect of respiratory syncytial virus on subsequent allergic sensitization to ovalbumin in guinea-pigs.

Children with acute respiratory syncytial virus (RSV) bronchiolitis often develop recurrent wheezing, asthma and allergic sensitization, but the role of RSV in the pathogenesis of these sequelae is unclear. This study examined whether RSV infection potentiates subsequent allergic sensitization, airway hyperresponsiveness (AHR) and airway inflammation induced by repeated exposures to aerosolized ovalbumin (OA) in guinea-pigs. Guinea-pigs received either RSV or sham inoculum, followed by exposures to OA- or saline-containing aerosols to form the following groups: 1) noninfected, nonsensitized controls (sham/saline group); 2) RSV-infected, nonsensitized animals (RSV/ saline group); 3) noninfected, OA-sensitized animals (sham/OA group); 4) RSV infection and first OA exposure on the same day (RSV/OA group), and 5) RSV infection six days prior to first OA exposure (RSV6/OA group). Three days after the final aerosol exposure, circulating OA-specific immunoglobulin (Ig)G1 antibody titres and AHR to inhalation acetylcholine challenge were measured and morphometry performed to evaluate allergic inflammation of the airways. OA-exposed animals developed OA-specific IgG1 antibodies, AHR and airway eosinophilia (sham/OA, RSV/OA and RSV6/OA groups. RSV infection alone induced significant AHR and airway eosinophilia (RSV/saline group). RSV infection, and concomitant exposure to OA (RSV/OA group) enhanced OA-specific IgG1 antibodies, but not airway eosinophilia or AHR. Such increases were not observed in the RSV6/OA group. In conclusion, respiratory syncytial virus potentiates the production of ovalbumin-specific immunoglobulin G1 antibodies in guinea-pigs, but circulating titres of these antibodies do not reflect the extent of airway hyperresponsiveness or airway inflammation. In addition, respiratory syncytial virus infection alone can produce slight increases in airway hyperresponsiveness that are associated with increased numbers of eosinophils in the airways.

Allergens↗

Respiratory syncytial virus infection and virus-induced inflammation are modified by contaminants of indoor air.

The airway epithelium is the first cellular component of the lung to be encountered by the particles and pathogens present in inhaled air. In addition to its role as a physical barrier, the immunological activity of the airway epithelium is an essential part of the pulmonary immune system. This means that the symptoms of lung diseases that involve immunological mechanisms are frequently exacerbated by infection of the airway epithelium with respiratory viruses. The virus-induced enhancement of immunological activity in infected epithelial cells is well characterized. However, the effects that contaminants of inhaled air have upon the infectivity and replication of respiratory viruses and the inflammation they cause, are comparatively unknown. In this study, we have shown that pre-exposure of airway epithelial cells to bacterial lipopolysaccharides or a proteolytically active house dust mite allergen, is able to, respectively, inhibit or enhance the level of cellular infection with respiratory syncytial virus and similarly alter virus-induced expression of the inflammatory chemokine interleukin-8. These results suggest that respiratory syncytial virus infection and the inflammation caused by respiratory syncytial virus may be modified by the biologically active contaminants of indoor air.

Air Pollutants↗

The impact of influenza viruses on hospitalizations in infants younger than two years old during epidemics of respiratory syncytial virus infection.

In order to evaluate the association of influenza viruses with hospitalizations for acute respiratory infection in infants younger than two years old during epidemics of respiratory syncytial virus infection, we studied 512 nasal washes from this population. The samples were obtained from 1997 to 2000. A total of 337 viruses were isolated: 264 respiratory syncytial viruses, 62 influenza viruses, eight parainfluenza viruses, two adenovirus and one rhinovirus. Hospitalizations for acute respiratory infection were owing to influenza and respiratory syncytial viruses in 18.3% vs. 78.3% of all cases, and 32.5% vs. 65.8%, respectively, in the group of infants between 6 months and 2 years old.

Hospitalization↗

Alpha-2A-interferon for treatment of bronchiolitis caused by respiratory syncytial virus.

Alpha-2a-interferon (IFN) has been shown to provide important local defense against some viral infections. In vitro, IFN inhibits the growth of respiratory syncytial virus in bovine monolayer cultures, but respiratory syncytial virus is known to be a poor inducer of IFN in infected infants. When IFN was administered by daily intramuscular injection in a double blind, placebo-controlled study of 22 infants with bronchiolitis caused by respiratory syncytial virus, there was no statistically significant difference in clinical course, duration of oxygen requirement or physical assessment between the treatment and control groups. Likewise viral isolation showed no difference between the groups. Further studies are needed to determine whether higher dosing or aerosol or other alternative route of delivery of IFN might lead to therapeutic benefit.

Bronchiolitis, Viral↗

Respiratory syncytial virus vaccine development.

Respiratory syncytial virus is the major respiratory pathogen of infants and children worldwide. Currently, there is no effective vaccine to protect against respiratory syncytial virus infection. Immunoprophylaxis with hyperimmune globulin or with a humanized monoclonal antibody is expensive, limited to children with underlying disease, and not practical for general use. Antiviral therapy is controversial and of limited effectiveness. New approaches to the development of a vaccine for respiratory syncytial virus infection are promising. Several subunit vaccines and live attenuated virus vaccines are immunogenic and safe in children and adults. This review focuses on potential vaccine candidates and the challenges these candidate vaccines must overcome.

Adult↗

Impact of severe disease caused by respiratory syncytial virus in children living in developed countries.

Among industrialized nations, the rate of rehospitalization in the United States for respiratory syncytial virus (RSV) is approximately 30 per 1000, exceptions being noted for American Indians and Alaskan natives, two ethnic groups who tend toward higher rates of RSV hospitalization. In distinction Japan reports an admission rate of 60 per 1000 for RSV disease. Yet Japan ranks considerably lower than many of its western counterparts in premature births. Whether an RSV subtype, a new viral genotype or some other unifying characteristic exists that might explain the severity of adenovirus, parainfluenza and RSV infections in this region of Asia remains to be determined. Outcomes trials in the United States, Canada, United Kingdom, Denmark and Japan all identified crowding and exposure to tobacco smoke as significant and independent risk factors for disease severity of RSV. The epidemiology of RSV is largely consistent throughout Europe, with peak outbreaks occurring in December and January. In Europe RSV accounts for 42 to 45% of hospital admissions for lower respiratory tract infections in children younger than 2 years of age, and inpatient populations tend to be younger and to experience greater disease severity. For RSV bronchiolitis lengths of stay in European hospitals range from a low of 4 days to a high of 10 days. The Infección Respiratoria Infantil por Virus Respiratorio Sincitial Study Group in Spain conducted 2 prospective observational studies in 14 and 26 neonatal units, respectively, on nonprophylaxed neonates to determine hospitalization rates for respiratory syncytial viral illness during 2 consecutive RSV seasons. Throughout each respiratory season the study group followed premature infants of < or =32 weeks gestational age at birth, representing an annual birth cohort of approximately 100 000 infants. A total of 584 infants who were < or =32 weeks gestational age in the first season and 999 in the second season were followed at monthly intervals. The nonprophylaxed hospitalized group was compared with the nonprophylaxed, nonhospitalized group, and presumptive risk factors were determined for rehospitalization among premature infants. These independent variables, similar for both years, were identified as low gestational age, underlying chronic lung disease, living with school age siblings, exposure to tobacco smoke and a chronologic age of < 3 months at the onset of the RSV season.Stable, yet high rates of admission for RSV illness in Spain were observed in this premature group of < or =32 weeks gestational age: 13.4% for 1999; and 13.1% for the year 2000. Of those hospitalized during the 2 years of the study, 18 and 25%, respectively, were admitted to the intensive care unit. With the exception of higher rates of family allergy, multiple deliveries and a lower rate of neonatal morbidity, prognostic variables for high risk of hospital admission in the year 2000 compared with those of the 1999 sample. Findings from this comprehensive, prospective study served as the basis for the development of standards for the prevention of respiratory syncytial virus infection by the Spanish Society of Neonatology.

Europe↗

Nucleotide sequence analysis of the non-structural NS1 (1C) and NS2 (1B) protein genes of bovine respiratory syncytial virus.

The nucleotide and deduced amino acid sequences of the non-structural (NS) protein genes of bovine respiratory syncytial virus (BRSV) strain A51908 were determined. The NS1 and NS2 genes were 524 and 489 nucleotides long with single open reading frames, encoding polypeptides of 136 and 124 amino acids, respectively. Comparison of the NS1 gene of BRSV with the corresponding sequences of ovine respiratory syncytial virus (ORSV) and human respiratory syncytial virus (HRSV) subgroups A and B revealed 82, 67 and 65% identity at the nucleotide level, and 89, 69 and 68% identity at the amino acid level, respectively. The nucleotide identity of the NS2 gene of BRSV to those of ORSV and HRSV subgroups A and B was 80, 69 and 67% and the predicted amino acid identity was 87, 84 and 83%, respectively. The intergenic sequences of the NS1-NS2 and NS2-N gene junctions of BRSV were determined and found to be 13 nucleotides shorter and 29 nucleotides longer, respectively, than the corresponding regions of HRSV.

Amino Acid Sequence↗

Rapid detection and clinical features of infants and young children with acute lower respiratory tract infection due to respiratory syncytial virus.

During December to the end of February of 2003 and 2004, a total of 282 nasopharyngeal aspirates were obtained from infants and young children admitted to the Buraidah Maternity and Pediatric Hospital, Al-Qassim, Saudi Arabia, and clinically diagnosed as suffering from acute lower respiratory tract infections. The aspirates were tested for the presence of respiratory syncytial virus using direct fluorescein-labeled monoclonal antibody assay. Of the 282 specimens, 128 (45.4%) were found to be positive for respiratory syncytial virus. The most positive specimens came from patients less than one year old (51.3%), and were associated with bronchopneumonia (56.7%) or bronchiolits (55.4%). Coughing (100%) and tachpnea (98%) were significantly more frequent in infants with respiratory syncytial virus infection, followed by wheezing, crepitation and retraction, each representing 66%. Three deaths were reported. The availability of a rapid viral diagnostic assay will be an important tool for physicians to make more accurate treatment decisions and therefore reduce unnecessary antibiotic usage and hospital stay for the patients.

Age Factors↗

Safety and pharmacokinetics of vitamin A therapy for infants with respiratory syncytial virus infections.

Infants with respiratory syncytial virus infection have low serum vitamin A levels. We treated 21 respiratory syncytial virus-infected children with 12,500 to 25,000 IU of oral vitamin A. Vitamin A levels were normalized at 6 h, and none of the children experienced vitamin A toxicity or exacerbation of respiratory illness. Vitamin A treatment of previously healthy respiratory syncytial virus-infected infants at these doses is safe and well tolerated.

Humans↗

Infected cell types in ovine lung following exposure to bovine respiratory syncytial virus.

Sixteen adult sheep (ten females, six males obtained from a closed flock at National Animal Disease Center, Ames, IA) were experimentally infected with bovine respiratory syncytial virus strain 375 (BRSV), and lung tissues were stained for viral antigen. Two infected sheep were euthanatized at each of the following post-inoculation times: 12, 24, 36, 48, 72, 96, 144, and 192 hours. Lung, nasal turbinates, trachea, right cranial bronchial and mediastinal lymph nodes, liver, and spleen were collected for histologic evaluation. An indirect immunoperoxidase technique was performed on routine paraffin-embedded sections of lung tissue, trachea, turbinates, and bronchial and mediastinal lymph nodes to determine the location of the BRSV antigen. For lung tissue from each sheep 400 light microscopic fields at 160x magnification were examined for staining for BRSV antigen. Lung tissue was also collected for virus and bacterial isolation. Daily serum samples were taken for determination of anti-BRSV titers. Severe respiratory disease was not produced in any sheep. Bovine respiratory syncytial virus was isolated from lung tissue collected from all sheep up through 144 hours post-inoculation. At 12 hours post-inoculation (case No. 2) respiratory syncytial virus antigen was detected in bronchiolar epithelium and a mononuclear cell within an alveolar space. Lung tissue from the sheep necropsied between 24 and 144 hours post-inoculation (case Nos. 3-14) contained BRSV antigen in bronchiolar epithelium, type I pneumocytes, type II pneumocytes, alveolar macrophages, and mononuclear cells within alveolar spaces. Macrophages staining for viral antigen were rare. Bronchiolar and type I epithelial cells comprised the majority of infected cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗