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Biomedical subjects

H O Kangro

Publications and source records attributed to H O Kangro.

At least 19 recordsLinked to original sources
PubMed1992

Epstein Barr virus (EBV) encoded small RNAs: targets for detection by in situ hybridisation with oligonucleotide probes.

AIMS: To develop a rapid, sensitive, and specific non-isotopic in situ hybridisation (NISH) procedure for the detection of Epstein-Barr virus in formalin fixed, paraffin wax embedded tissues. METHODS: Two low molecular weight RNAs, designated EBER-1 and EBER-2 (Epstein-Barr encoded RNA), were used: cells latently infected with EBV secrete large amounts of EBERs. The method uses digoxigenin labelled anti-sense oligonucleotides, corresponding to sequences in EBER-1 and EBER-2. RESULTS: The use of these probes, in conjunction with high temperature microwave denaturation, ensured that the technique was considerably more sensitive than other in situ hybridisation techniques for detecting EBV. Furthermore, the hybridisation signal was morphologically distinct in that only the nucleus and not the nucleolus give a positive signal. No cross-hybridisation was observed with cells infected with other lymphotropic herpes viruses. CONCLUSION: The sensitivity, simplicity, and rapidity of this technique make it ideal for diagnostic use, and for studies investigating the role of this virus in neoplastic disease.

Cell Line↗
PubMed1992

Presence of Epstein-Barr virus in Hodgkin's disease is not exclusive to Reed-Sternberg cells.

Thirty-three cases of Hodgkin's disease (HD) have been studied for the presence of Epstein-Barr virus (EBV) using a novel nonisotopic in situ hybridization procedure, based on the detection of Epstein-Barr encoded RNAs with oligonucleotide probes. An intense and morphologically distinct nuclear staining, sparing the nucleolus was seen in a total of 12 cases (36%). In six of these cases, the signal was located to the Hodgkin and Reed-Sternberg cells (HR-S); in the other six positive cases, the signal was observed only in the non-neoplastic small lymphocytes. These lymphocytes were few in number and immunocytochemistry results were consistent with a B-cell phenotype. The presence of EBV in those cases characterized by nuclear staining of small lymphocytes was confirmed by the polymerase chain reaction (PCR) analysis. The authors report the detection of EBV in small lymphocytes in HD by in situ hybridization and discuss the implications of these findings in relation to the proposed etiologic association between EBV and HD.

Adolescent↗
PubMed1991

Antibody avidity following varicella-zoster virus infections.

The avidity of IgG antibodies following varicella-zoster virus (VZV) infections was investigated using urea treatment of antigen-bound serum antibody by indirect radioimmunoassay (RIA) and immunoblotting techniques. Sequential sera from 16 patients with varicella and 17 patients with zoster were tested, as well as sera from 80 seropositive individuals without a recent history of VZV disease. Both types of assay showed that low-avidity antibodies predominate early after primary infection, but that antibody avidity increases markedly during convalescence. Using RIA, all sera taken up to 12 weeks after the onset of varicella showed greater than 50% reduction in antibody titre after treatment with 8 M urea but thereafter the proportion of urea resistant antibody increased with time. In contrast, after recurrent infection, high avidity antibodies were found to predominate at all times. Only 6 of 47 sera tested from zoster cases showed greater than 30% reduction after urea treatment and all these were taken within 2 weeks after onset of rash. Immunoblotting also showed that the highly immunogenic p32/p36 nucleoproteins appear to induce predominantly low avidity antibodies, even after recurrent VZV infection. The results of this study indicate that treatment with 8 M urea in RIA for IgG antibodies may be a simple and reliable method for distinguishing primary and anamnestic antibody responses against VZV.

Antibodies, Viral↗
PubMed1991

Simplified procedures for applying the polymerase chain reaction to routinely fixed paraffin wax sections.

The polymerase chain reaction was applied to the analysis of DNA contained in archival paraffin wax embedded material. DNA suitable for the reaction was obtained from these tissues by simple extraction methods, without previous dewaxing of tissue sections. When compared with unfixed material, the reaction efficiency was compromised, so that an increased number of amplification cycles were required to produce equivalent amounts of amplified product. This in turn led to an increase in amplification artefacts, which can be minimised by a simple modification of the standard reaction. Amplification of relatively large DNA fragments was not always successful, and it seems prudent to bear this in mind when designing oligonucleotide primers which are to be used for the amplification of archival material. The efficiency of the procedure can be improved by dividing the amplification cycles into two parts: this reduces the amount of reagent needed, is relatively simple and inexpensive, and can be performed in one working day.

Base Sequence↗
PubMed1991

Inhibitory effects of urine on the polymerase chain reaction for cytomegalovirus DNA.

The inhibitory effects of urine samples taken from neonates and older children, some of which were known to be infected with cytomegalovirus, on the polymerase chain reaction (PCR) were investigated. Urea was the major inhibitory component of urine and inhibited the PCR at a concentration of more than 50 mM. Urine samples from older children were more inhibitory than those from neonates. This correlated with the higher concentration of urea generally found in urine samples from older children compared with neonatal urines. Two of 13 neonatal urine samples, however, were inhibitory despite low urea concentrations--presumably due to metabolites derived from parenteral nutrition. The inhibitory effects of urine were effectively removed by simple dialysis or ultrafiltration. The sensitivity and specificity of PCR for detecting cytomegalovirus DNA in urine were further improved by using "nested" primers and a modified PCR protocol entailing the use of reduced reactants in the first 20 cycles of a two-stage 50 cycle PCR.

Age Factors↗
PubMed1990

Antibody responses in recipients of varicella vaccine assayed by immunoblotting.

Sera taken from 35 children with cancer who had been vaccinated with live varicella vaccine were assayed using immunoblotting for the presence of IgG class antibodies to proteins present in varicella-zoster virus (VZV)-infected cells. Sera from 23 of these patients were also assayed for IgM class antibodies. The patterns of immunoreactivity observed for these patients following vaccination were substantially weaker and more variable than those detected following natural VZV infection in otherwise healthy individuals. The IgG responses detected following vaccination involved up to 10 protein bands between 28 and 188 kDa. Bands were particularly frequent in the 78-96 kDa region. IgM responses involved up to 10 bands between 28 and 114 kDa, with the bands in the 78-96 kDa region and at 32-36 kDa being detected most frequently. Repeated vaccination generally produced a stronger IgG antibody response than did single vaccination, and subsequent exposure of vaccinees to natural VZV infection resulted in an increased level of reactivity for IgG antibodies, but not for IgM. Similar reaction patterns were obtained with sera from vaccinees when the vaccine virus and wild-type VZV were used as antigens. Immunoblotting showed good correlation with indirect radioimmunoassay for the detection of a vaccine-induced IgG response.

Antibodies, Viral↗
PubMed1990

A prospective study of viral and mycoplasma infections in chronic inflammatory bowel disease.

Seventy-two children with chronic inflammatory bowel disease were investigated for infections with various viruses and Mycoplasma pneumoniae, Chlamydia psittaci, and Coxiella burnetii to determine whether these pathogens are associated with acute onset exacerbations. Altogether 54 infections were identified serologically, of which 23 (42.6%) were associated with exacerbations. This corresponded to 24.2% of the recorded exacerbations during the study period. The respiratory pathogens accounted for 59.3% of the infections and 43.8% of these were associated with gastrointestinal symptoms. This is consistent with the observation that up to 40% of the exacerbations were associated with symptoms of antecedent or concurrent infection, most commonly involving the respiratory tract. Rubella virus, Epstein-Barr virus, and adenovirus were associated with acute exacerbations in 5 children. Thus, common pathogens were frequently associated with exacerbations and account for a large proportion of the commonly reported symptoms of a concurrent infection. The possible causal relationship between these pathogens and exacerbation of inflammatory bowel disease is discussed. Reactivation of latent herpesviruses was identified in 4 children with active disease and indicates that the converse relationship may also occur.

Acute Disease↗
PubMed1990

Protein blotting: ten years on.

Protein blotting was originally described in 1979 as an outgrowth of nucleic acid techniques, and received its commonly used designation of 'Western' blotting in 1981. The use of the technique to render electrophoresed proteins accessible for further analysis has found many roles, the most prominent being subsequent reaction with antibodies or antisera, which has many clinical and research applications. Since the initial development of the system there have been many changes to the techniques involved, but the basic principles remain unaltered. This review discusses these changes, and also provides a summary of current techniques.

Antibodies↗
PubMed1990

Lipoproteins of varicella-zoster virus.

Human fibroblast cells infected with varicella-zoster virus (VZV) showed a slight increase in lipoprotein synthesis, with the production of two major viral lipoproteins, as detected by radioimmunoprecipitation (RIP). Three bands of Mr 73,000, 90,000 and 97,000 were identified as forms of the VZV gpI glycoprotein. All three incorporated both palmitic and myristic acid, and were shown by thin-layer chromatography to contain myristic, palmitic and stearic acids. A very strong band corresponding to 7,000 Mr, which may represent the product of VZV gene 49, was detected after RIP and in VZV-infected cells, and was shown to contain almost entirely myristic acid. Several minor bands were also detected. The possible functions of the lipoproteins are discussed.

Cell Line↗
PubMed1989

Discordant results obtained on testing sera from immunocompromised patients for cytomegalovirus IgG by enzyme-linked immunosorbent assay and radioimmunoassay.

The investigation of sera from immunocompromised patients for antibody to CMV by ELISA, RIA, immunofluorescence (IF) and complement-fixation (CF) revealed discrepancies that reflected differences in test specificity rather than sensitivity and suggested that for the long-term serological follow-up of such patients it would be advisable not to rely on only a single assay procedure.

Adult↗
PubMed1988

Serological responses in varicella and zoster assayed by immunoblotting.

Acute and convalescent zoster sera taken from 11 patients with varicella and 12 patients with zoster were assayed using immunoblotting for the presence of IgG- and IgM-class antibodies to proteins present in varicella-zoster virus-infected cells. All patients exhibited a detectable virus-specific response with both antibody classes. The IgG responses involved up to 28 protein bands between 28 and 255 kilodaltons (kDa). The reactivity was particularly strong in the 78-114-kDa region, with additional bands observed with all patients at 32, 35, 66, and 220 kDa. This pattern of reactivity typically developed more slowly and was weaker and more variable in patients with varicella compared to those with zoster. The reactivity of IgM antibodies in immunoblotting was similar after varicella and after zoster. Individual sera showed up to 25 bands, with the major reactivity being directed against the 78-96-kDa region and two bands at 32 and 35 kDa. Some differences were apparent between the primary and anamnestic responses with both IgG and IgM antibodies, but this did not allow reliable discrimination of the two types of infection.

Adult↗
PubMed1988

Reduction in immunoreactivity of varicella-zoster virus proteins induced by mycoplasma contamination.

During the study of protein differences between several strains of varicella-zoster virus (VZV), two of the strains were found to be contaminated with Mycoplasma hyorhinis. Polyacrylamide gel electrophoresis showed fourteen extra bands present in MRC5 fibroblasts infected with these strains compared to other strains of VZV. A more striking difference was observed when infected cultures were used as antigens in immunoblotting. Certain viral proteins, corresponding in molecular weight to the viral glycoproteins, showed greatly reduced immunoreactivity. Experimental contamination of a mycoplasma-free strain of VZV with the glucose fermenting M. hyorhinis produced similar effects on immunoreactivity, while contamination with the arginine-hydrolysing M. orale produced no detectable effects. Given these data, it appears likely that the glucose-fermenting species induced significant changes in the VZV glycoproteins, possibly by depletion of sugars or interference in glycosylation pathways. The implications of this are discussed.

Cells, Cultured↗
PubMed1988

Detection of specific IgM in varicella and herpes zoster by antibody-capture radioimmunoassay.

A simple and sensitive M antibody-capture radioimmunoassay (MACRIA) is described which utilizes crude commercial VZV antigen and a single monoclonal anti-VZV antibody. This was compared to the immunofluorescence (IF) test for IgM antibody and was used to study IgM responses in sera from 261 patients with varicella and 220 patients with herpes zoster. With MACRIA, IgM antibodies were detected in all patients with varicella. The IgM antibodies appeared shortly after onset of rash, reached peak levels between 1 and 4 weeks after onset and then declined to low or undetectable levels in most, though not all, patients after 3 months. IgM antibodies were also detected in 98.2% of patients with herpes zoster, but the levels of IgM were significantly lower than after varicella and there was wider individual variation both in magnitude and duration of the IgM responses, in some cases only lasting 2-3 weeks. Comparison between MACRIA and IF showed good agreement in the detection of IgM antibodies following varicella. Discordant results were obtained with 13% of sera, of which 81% were taken either early or late after onset of rash and contained very low IgM levels. In contrast, 62 (28%) of the 220 sera from patients with zoster gave discordant results in the two tests, all except five being MACRIA-positive but IF-negative. The largest proportion of discordant results were obtained with sera taken more than 3 months after onset of rash, but 18 (29%) contained high IgM levels and were taken during the period of peak IgM responses. The diagnostic applications of the VZV MACRIA are discussed.

Chickenpox↗
PubMed1987

Efficacy of varicella vaccine in patients with solid tumours.

Thirty nine children with malignant disease and without antibodies to varicella zoster virus were immunised with a live Oka strain varicella vaccine. Seroconversion was shown in 24 of those who were vaccinated but five of 18 who responded to the vaccine who were followed up for over six months subsequently lost their antibodies. Of 10 children who were revaccinated, nine responded to the second dose but three lost their antibodies within six months to two years. Four children developed reactions related to the vaccine, one local and three generalised, but all these were transitory and of no clinical concern. Nine of those who were vaccinated, including four who did not respond to the vaccine, have subsequently had close exposure to varicella but none has developed the disease.

Antibodies, Viral↗
PubMed1986

The effect of staining on the immunoreactivity of nitrocellulose bound proteins.

It was found that Coomassie blue staining of proteins electroblotted onto nitrocellulose resulted in a significant decrease in subsequent immunoreactivity, relative to unstained proteins. This was not uniform, and resulted in distortion of the pattern detected by immunoreaction as well as an overall decrease in sensitivity. Staining with amido black resulted in no such alteration. Additionally, it was observed that amido black is completely leached from the nitrocellulose strip during immunoreaction, whereas no leaching is apparent with Coomassie blue. This may contribute to the observed effect of staining on immunoreactivity.

Antigen-Antibody Reactions↗