Biomedical subjects

M Waris

Publications and source records attributed to M Waris.

At least 19 recordsLinked to original sources

PubMed1999

Simultaneous detection of IFN-gamma and IL-4 mRNAs using RT-PCR and time-resolved fluorometry.

Time-resolved fluorometry was applied in the detection of RT-PCR amplified mRNAs for the Th1 and Th2 cell-derived cytokines interferon gamma (IFN-gamma) and interleukin (IL-)4, respectively. RNA stimulated cells was reverse transcribed and the cDNAs for the cytokine mRNAs and the constantly expressed beta-actin (beta-ACT) mRNA were simultaneously amplified in one multiplex PCR reaction. The PCR conditions were optimized to minimize mutual inhibition of individual amplifications. One of the PCR primers in each primer pair was biotinylated, and the PCR products were captured onto streptavidin-coated microtitre plates. The three PCR products were detected with three different lanthanide labelled target-specific probes in solution hybridization. IFN-gamma, IL-4 and beta-ACT were detected with europium (Eu), terbium (Tb) and samarium (Sm) labelled probes, respectively, using time-resolved fluorometry. Small cell numbers used in microtitre plate cultures were sufficient to detect cytokine messages after mitogen stimulation. This sequence-based method provides a sensitive, specific, fast and nonisotopic alternative to conventional blotting and hybridisation with radioactive probes. In addition, the multiplex fluorogenic dye detection facilitates relative quantification of target mRNAs.

Actins↗

PubMed1998

Development of human antibodies against linear antigenic and immunogenic regions of respiratory syncytial virus (RSV) nucleocapsid and phospho-proteins shows the site-directed characteristics.

BACKGROUND: The successful development of an RSV vaccine requires a better understanding of the pathogenesis of primary infection, susceptibility to reinfection, and the immunopathology of enhanced illness in children immunized with a non-replicating RSV candidate vaccine. The exact role of different immune parameters in RSV pathogenesis remains controversial. OBJECTIVES: To study the contribution of antibodies directed to the linear antigenic and immunogenic regions of the N and P proteins in the titer rise and avidity maturation of total anti-RSV antibodies. STUDY DESIGN: The occurrence of antibodies directed against three linear antigenic and immunogenic regions in each of the nucleocapsid (N): N3 (Thr11 to Gly30), N25 (Ser231 to Ala250) and N39 (Thr371 to Leu391), and the phospho-(P) proteins of respiratory syncytial virus (RSV), subgroup A: P49 (Pro91 to Asp110), P56 (Ser161 to Lys180) and P62 (Glu221 to Phe241), were analyzed in ELISA with (a) 32 paired sera from humans with recent or previous RSV subgroup A and/or B infection diagnosed by conventional ELISA, detection of antigen in nasopharyngeal aspirates and measurement of antibody avidity change; and (b) 40 RSV antibody-positive sera (HCS) obtained from patients during their convalescence from RSV infection and possessing clearly demonstrable titers of RSV IgG in conventional enzyme immunoassays (EIA) based on whole RSV antigen. RESULTS: The titer rise of antibodies directed to the combined three peptides representing the RSV N protein was well correlated with the rise in anti-RSV antibodies measured in whole antigen ELISA. Surprisingly, the rise in antibodies against a truncated main C-terminal epitope (Gln381 to Leu391) of the N protein (represented by subgroup A specific sequence of the N39/1 peptide) was inversely correlated with the titer rise of total anti-RSV antibodies. The titer rise of antibodies to the C-terminal linear site of the RSV N (N39/1) protein was subgroup-specific during the course of primary RSV infection. A titer rise in antibodies to the C-terminal linear sites of RSV N (i.e. N39/1) and P (P62) proteins had a dominating appearance in sera from newborn infants (6-7 months) and from patients with RSV reinfections. Anti-RSV antibody titers of late convalescent sera correlated with the titers of antibodies directed to the C-terminal linear site of RSV P (P62) protein. The avidity maturation of the anti-RSV immune response followed the titer rise of anti-P62 antibodies during the course of primary or secondary RSV infection.

Antibodies, Monoclonal↗

PubMed1998

Intranasally administered immunoglobulin for the prevention of rhinitis in children.

OBJECTIVE: To determine the efficacy of intranasally administered immunoglobulin in preventing symptoms of rhinitis in children. METHODS: Forty children ages 1 to 4 years who attended day-care centers in Turku, Finland, were enrolled in the double blind, placebo-controlled study. The children were randomly assigned to receive treatment with immunoglobulin, composed mainly of immunoglobulin A, or placebo, both administered as nasal sprays twice daily for 8 weeks. During this medication period and an additional 8-week follow-up period, the parents recorded the symptoms of the children daily in the diaries provided. One child who met an exclusion criterion was withdrawn from the study after a few days of medication. RESULTS: During the 8-week medication period the 19 children in the immunoglobulin group had 42% fewer days with rhinitis than the 20 children receiving placebo (mean, 10.8 vs. 18.7 days; P=0.004). The total numbers of episodes of rhinitis in the immunoglobulin and placebo groups were 33 and 51, respectively. No significant differences were observed between the groups during the postmedication follow-up period. CONCLUSIONS: Intranasal administration of immunoglobulin appears to be an effective method to prevent symptoms of rhinitis in children, and further studies of this approach are needed.

Administration, Intranasal↗

PubMed1998

A new method for automatic wheeze detection.

A new automatic wheeze detection method which is based on image processing techniques applied to the sonagram was developed here. In the calculation of the sonagram, autoregressive and FFT spectrum estimation methods were compared. The method was validated in four wheezing asthmatic patients by a pulmonary physician. Nine out of ten wheezes longer than 250 ms were detected. Very short wheezes were not detected. The false positive amount of wheezing in control subjects was only about 1%. The method extracts also information about the frequency, duration, flow and volume associated with the wheezes.

Asthma↗

PubMed1995

Preparation of monoclonal antibodies for the diagnosis of influenza A infection using different immunization protocols.

Four immunization protocols were used to obtain cross-reactive influenza type A-specific monoclonal antibodies: (1) repeated administration of purified influenza virus, (2) immunization with bromelain-treated viral particles free of HA and NA, (3) sequential immunization with two strains of different subtypes, and (4) immunization with bromelain-treated particles following tolerization of mice to surface glycoproteins by cyclophosphamide. The fourth approach was shown to be the most effective since a high proportion of hybridomas producing cross-reactive influenza virus type A-specific MAbs were obtained. MAbs of type A specificity were immunochemically characterized and examined for their ability to detect virus in clinical specimens. It was demonstrated that two pairs of the newly prepared MAbs provided excellent reagents for viral detection in clinical specimens using time-resolved fluoroimmunoassay.

Animals↗

PubMed1995

Inhibition of herpes simplex virus type 1, respiratory syncytial virus and echovirus type 11 by peroxidase-generated hypothiocyanite.

The human mouth is an important route of viral transmission and evidence exists that human saliva can neutralize some viruses, e.g. herpes simplex type 1 (HSV-1) and human immunodeficiency virus (HIV) in vitro. However, little is known of the actual antiviral agents in saliva. We have analyzed how hypothiocyanite (HOSCN/-OSCN) ions, present in human saliva and generated by salivary peroxidase systems, affect the viability of three different types of viruses; HSV-1 (capable of inducing oral lesions), respiratory syncytial virus (RSV, respiratory infections), and echovirus 11 (EV 11, enteric diseases). Viral suspensions were pretreated (30 min) with HOSCN/-OSCN concentrations up to 180 microM both at pH 6.0 and 7.1 and inoculated into human gingival fibroblasts. The cultures were incubated at 37 degrees C for 18-48 h, fixed and the infected cells were counted after immunoperoxidase staining. HSV-1 was most sensitive to HOSCN/-OSCN with an IC50 of 8.5 microM at pH 6.0 and an IC50 of 20 microM at pH 7.1, respectively. RSV was inhibited by HOSCN/-OSCN only at pH 6.0 with an IC50 of 8.0 microM. EV 11 was also resistant at neutral pH, but sensitive at pH 6.0 with an IC50 of 68 microM. In contrast to HSV-1 and RSV, the inhibition of EV 11 was not dependent on the concentration of HOSCN/-OSCN. The inhibition was in all cases stronger at pH 6.0 than at neutral pH. Our results suggest that hypothiocyanite, a normal component of human whole saliva, in physiological concentrations effectively inhibits HSV-1 and RSV at acidic pH, whereas EV 11 is more resistant in vitro.

Adult↗

PubMed1995

Short-term use of amoxicillin-clavulanate during upper respiratory tract infection for prevention of acute otitis media.

We performed a randomized, double-blind, placebo-controlled study to determine whether acute otitis media could be prevented by antibiotic therapy initiated promptly after the appearance of symptoms of upper respiratory tract infection. One hundred four children aged 1 to 4 years received a 7-day course of either amoxicillin-clavulanate or placebo. Acute otitis media developed in 9 (18%) of the 50 children receiving amoxicillin clavulanate and in 12 (22%) of the 54 children receiving placebo (p = 0.59).

Acute Disease↗

PubMed1995

Linear antigenic and immunogenic regions of human respiratory syncytial virus N protein.

Three linear antigenic regions on the N protein from human respiratory syncytial virus (RSV) subgroup A (strain A2) were identified by using peptides which reacted in ELISA with sera from humans with recent or previous RSV infection. The determinants were localized within three hydrophilic regions of the protein: Thr11 to Gly30 (N3 peptide), Ser231 to Ala250 (N25 peptide) and Thr371 to Leu391 (N39 peptide). The site represented by the N39 peptide reacted with four subgroup A-specific MAbs. There were minor variations in the amino acid epitope dependencies of each of these MAbs. Two additional antigenic regions Ser131 to Arg150 and Ala181 to Leu200, were represented by peptides that reacted with human convalescent sera, but these peptides did not differentiate between acute and convalescent sera from RSV-infected humans. Rabbit hyperimmune sera raised against selected peptides specifically precipitated different forms of the N protein from a nucleocapsid-containing homogenate derived from extracts of RSV-(subgroup A and/or B)-infected 35S-labelled cells in a radioimmuneprecipitation assay (RIPA); the sera were also used to demonstrate RSV infection in cells by immunofluorescent assay (IFA). Anti-N3 peptide sera precipitated N41, the full-length (M(r) 41,000) form of N protein, in a RIPA done on a soluble protein pool. Anti-N39 (C-terminal region) peptide sera precipitated both forms, suggesting that N38 (M(r) 38,000) is an N-terminally truncated (probably at position Tyr23 located inside the N3 peptide linear antigenic region) form of N41 protein.

Amino Acid Sequence↗

PubMed1995

Incidence of acute otitis media associated with group A and B respiratory syncytial virus infections.

The comparative association of respiratory syncytial virus group A and B infections with acute otitis media was determined by analysing the hospital records of children with community-acquired respiratory syncytial virus infection during three successive outbreaks from 1987 to 1992. Of 326 episodes analysed, 192 (59%) were caused by group A and 134 (41%) by group B infections. Acute otitis media was diagnosed in 101 (75%) children with group B infection, compared with 119 (62%) with group A infection (p = 0.01). Group A infections were more often associated with wheezing (71% versus 59% in group B; p = 0.02) and oxygen therapy in inpatients (48% versus 31%, respectively; p = 0.008). The higher incidence of acute otitis media associated with group B infections was observed both after adjustment for potential confounding variables and during each outbreak.

Acute Disease↗

PubMed1994

Linear antigenic and immunogenic regions of the respiratory syncytial virus P protein.

Three linear antigenic regions on the P protein from human respiratory syncytial virus (RSV) subgroup A (strain A2) were represented by peptides that reacted with monoclonal antibodies and with sera from humans with recent or previous RSV infection. The determinants were localized within three hydrophilic regions of the P protein: Pro91 to Asp110, Ser161 to Lys180 and Glu221 to Phe241. The role of individual amino acids in the epitopes defined by monoclonal antibodies was determined. Two monoclonal antibodies reacting with the same antigenic site were found to detect epitopes that had different amino acid dependencies. Rabbit hyperimmune sera raised against selected peptides specifically precipitated different forms of the P protein from RSV-infected 35S-labelled cell extracts in a radioimmune precipitation assay. These findings have implications for forthcoming structural-functional studies of RSV capsid component interactions and also for serological diagnosis of RSV infection.

Amino Acid Sequence↗

PubMed1992

Shedding of infectious virus and virus antigen during acute infection with respiratory syncytial virus.

Shedding of respiratory syncytial virus (RSV) in nasopharyngeal aspirates (NPA) of hospitalized children with acute respiratory infection was studied using direct antigen detection by time-resolved fluoroimmunoassay, rapid identification of infectious virus in centrifugally inoculated cell cultures by immunoperoxidase staining and conventional virus culture. Sequential NPAs, in which also local RSV-specific IgA response was measured, were collected from children with proven RSV infection. The shedding pattern was similar for both infectious virus and viral antigen. The overall agreement of the three methods was good (81%) in diagnostic specimens collected on admission, but markedly reduced (46%) in follow-up specimens. Secretory IgA was abundant in specimens giving discrepant or negative results only. The proportion of patients who shed RSV was high (> or = 87%) in the first week after onset of symptoms, and decreased sharply in the second week. An opposite temporal pattern was found in the proportion of patients with detectable RSV-IgA in their secretions. Sequentially isolated strains were antigenically stable as determined by their reactivity with a large panel of monoclonal antibodies. The findings suggest that RSV shedding should be monitored by using more than one method for virus detection.

Acute Disease↗

PubMed1992

Immunoglobulin G antibody avidity in patients with respiratory syncytial virus infection.

The titer and avidity of respiratory syncytial virus-specific antibodies were measured in 196 serum specimens from 93 children with an acute, laboratory-confirmed respiratory syncytial virus infection. An enzyme immunoassay method based on the ability of urea to dissociate the bound antibodies with low avidity from the antigen was used. Three patterns of immune responses were observed. Children less than 6 months of age usually had low titers of antibodies with high avidity in their acute-phase serum samples. These antibodies were concluded to be of maternal origin, since their reaction pattern was similar to that of healthy adults. During the next few weeks, a slight increase in titers with a concurrent decrease in antibody avidity was observed. All children 6 to 24 months of age had low-avidity antibodies in their acute-phase serum samples, which matured to high avidity during the follow-up. On the contrary, about half of the children greater than 24 months of age had high-avidity antibodies already in the acute-phase serum samples. We conclude that the former children were experiencing primary infections with respiratory syncytial virus and the latter were experiencing reinfections. All adults with remote immunity had antibodies with high avidity.

Adult↗

PubMed1991

Influenza vaccination in the prevention of acute otitis media in children.

We studied a new approach to the prevention of acute otitis media through the administration of influenza vaccine to 187 day-care center children aged 1 to 3 years before the influenza A epidemic of 1988-1989. The control group consisted of 187 unvaccinated children of similar age and background. During the 6-week study period, influenza A infection was diagnosed in five (3%) of 187 vaccinees and in 29 (16%) of 187 controls. Acute otitis media developed in three (60%) of five vaccinees with an influenza A infection compared with 18 (67%) of 27 controls (excluded were two children with a double viral infection). The incidence of acute otitis media associated with influenza A was reduced by 83% in the vaccinees. The total number of children with acute otitis media in the vaccine group was 35, compared with 55 in the control group, disclosing a 36% reduction among the vaccinees. We conclude that influenza vaccination decreases the incidence of acute otitis media in children during an influenza A epidemic, suggesting also that other vaccines against respiratory viruses may be an effective way to reduce the incidence of acute otitis media.

Causality↗

PubMed1991

Facile detection of anatoxin-a in algal material by thin-layer chromatography with Fast Black K salt.

A method for facile high-capacity screening of algal samples for anatoxin-a (ANTX-a), a potent neurotoxin of Anabaena flos-aquae, is presented. The method is based on in situ colour reaction of algal extracts containing ANTX-a on a thin-layer chromatographic plate with the diazonium reagent Fast Black K salt, and subsequent separation of the orange-red product. The product, shown to be a stable 3,3-dialkyltriazene, is derived from a reaction involving the aliphatic secondary amino group of ANTX-a. The detection limit for ANTX-a is 10 micrograms g-1 of lyophilized algal material, which is comparable to earlier methods using more complex instrumentation.

Bacterial Toxins↗

PubMed1991

Pattern of respiratory syncytial virus epidemics in Finland: two-year cycles with alternating prevalence of groups A and B.

Time-resolved fluoroimmunoassay with monoclonal antibodies distinguishing between respiratory syncytial virus (RSV) group A and B strains was used to analyze their prevalence in Finland during 1981-1990 among 3285 patients with laboratory diagnosis of RSV, most of them hospitalized. The group typing of antigens in 608 RSV-positive nasopharyngeal aspirates showed a regular alternation of group prevalence, following the cyclic occurrence of the virus. Group A predominated in 73%-90% of specimens from 1981-1982, 1985-1986, and 1989-1990, whereas group B predominated in 70%-100% of specimens from 1983-1984 and 1987-1988. The epidemiologic occurrence of verified reinfections in hospitalized children and the group typing results indicated that children greater than 6 months of age during the first infection were more resistant to severe reinfection with the homologous than with the heterologous group of virus. The study shows that group antigenic variation of RSV has a significant effect on the epidemiology of the virus.

Antibodies, Monoclonal↗

PubMed1990

Rapid detection of respiratory syncytial virus and influenza A virus in cell cultures by immunoperoxidase staining with monoclonal antibodies.

Peroxidase-labeled monoclonal antibodies against respiratory syncytial virus (RSV) and influenza A virus were used for immunoperoxidase staining (IPS) of cell cultures inoculated with nasopharyngeal aspirates. Cells were grown in 24-well plates, and specimens were inoculated by low-speed centrifugation. Cultures were incubated for 2 days at 37 degrees C and then fixed, stained, and observed by light microscopy. IPS was compared with standard virus isolation by using cultures of human diploid fibroblasts and Vero, HEp-2, and HeLa cell lines for RSV and Madin-Darby canine kidney cells for influenza A virus; these cultures were inoculated with specimens that were previously stored at -70 degrees C. Of 40 known RSV-positive specimens, 30 were found to be positive on reinoculation by both methods, and an additional 5 specimens were found to be positive by IPS only. Of 190 specimens tested for influenza A virus, 14 were positive by IPS and in tubes, and a further 8 specimens were positive by IPS only. IPS was also compared with direct detection of viral antigens in nasopharyngeal aspirates by a time-resolved fluoroimmunoassay (TR-FIA). Fresh nasopharyngeal aspirates were inoculated into human diploid fibroblasts and Madin-Darby canine kidney cells and tested for RSV and influenza A virus, respectively, by IPS. Of 110 specimens tested for RSV, 37 were positive in total, 32 were positive by IPS, and 33 were positive by TR-FIA. Of 150 specimens tested for influenza A virus, 39 were positive in total, 35 were positive by IPS, and 34 were positive by TR-FIA. IPS of cultures inoculated by centrifugation and incubated for 2 days is a sensitive method for the diagnosis of respiratory virus infections, and 24-well plates allow for the easy processing of a large number of specimens.

Animals↗

PubMed1989

One-incubation time-resolved fluoroimmunoassay based on monoclonal antibodies in detection of influenza A and B viruses directly in clinical specimens.

A new modified enzyme immunoassay screening method was developed for testing hybridoma cultures, so as to select antibodies useful for solid phase assays. Samples of hybridoma cultures were incubated for 1 h with purified nucleoprotein preparation in microtiter wells precoated with rabbit anti-influenza A or B immunoglobulin, followed by washing and addition of anti-mouse HRPO-conjugate. The monoclonal antibodies were then used in one-incubation time-resolved fluoroimmunoassay (TR-FIA) for detecting influenza viral proteins in nasopharyngeal aspirate specimens. The sample and europium (Eu)-labelled monoclonal detector antibody (100 microliter of each) were added simultaneously to microtiter wells precoated with anti-virus monoclonal antibody, and incubated for 1 h. After washing and addition of the enhancement solution the strips were shaken for 10 min before measurement of the fluorescence using a photon counting fluorometer. All of the monoclonal antibodies screened by our modified method and Eu-labelled worked as detector antibodies. Many of these monoclones also functioned as capture antibodies on solid phase. A total number of 309 (influenza A) and 104 (influenza B) nasopharyngeal aspirate specimens taken mainly from hospitalized children with acute respiratory disease were tested with the TR-FIA, using monoclonal antibodies produced by our modified screening method in comparison with monoclonal antibodies previously reported elsewhere (Walls et al., 1986). Results were similar, and superior to those obtained with routinely used indirect enzyme immunoassay based on polyclonal antibodies. The results of this study indicate that the one-incubation TR-FIA using monoclonal antibodies selected by the modified screening method is a highly sensitive and rapid method for detecting influenza A and influenza B virus in clinical specimens.

Antibodies, Monoclonal↗

PubMed1988

Herpes simplex virus detection by macroscopic reading after overnight incubation and immunoperoxidase staining.

Human diploid foreskin fibroblast cells grown in 24-well plates were inoculated with clinical specimens by centrifugation at 1,000 X g for 45 min. Cultures were incubated at 37 degrees C overnight, fixed, and stained with peroxidase-labeled monoclonal antibodies against herpes simplex virus types 1 and 2. Stained plaques of infected cells were large enough to be detected with the naked eye, and microscopic examination did not reveal any further positive specimens. The method was compared with standard isolation in human fibroblasts grown in shell vials and inoculated by centrifugation at 4,000 X g, observed microscopically for the occurrence of typical cytopathogenic effect three times a week for 10 days, and then typed by enzyme immunoassay. Of the 289 specimens tested, 105 were positive and 174 were negative by both methods. Six specimens were positive by standard isolation only, two of them containing varicella-zoster virus, and two specimens were stored frozen before being tested by immunoperoxidase staining. Two specimens found negative by standard isolation were positive by immunoperoxidase staining. For two specimens negative by immunoperoxidase staining, the standard isolation cultures were lost due to microbial contamination. Forty-two specimens found positive by standard isolation were clearly positive when stained only 8 h after inoculation. By standard isolation, positive results were reported on the average 3 to 4 days after inoculation, whereas by immunoperoxidase staining the result was available within less than 24 h. Immunoperoxidase staining of infected cells is a sensitive method for rapid laboratory diagnosis of herpes simplex virus infections, and 24-well plates are convenient for the handling of a large number of specimens.

Antibodies, Monoclonal↗