Activation and deactivation of membrane currents in human fibroblasts following infection with human cytomegalovirus.

The whole cell patch clamp technique was used to study the effects on membrane currents of infection of cultured human embryonic lung (HEL) fibroblasts with human cytomegalovirus (CMV). Four types of membrane currents were found in uninfected HEL cells, namely: Ca(2+)-activated potassium current, inward rectifier potassium current, delayed rectifier potassium current and voltage-dependent CMV. Voltage-dependent sodium current was detected in 30% of uninfected HEL cells whenever they were examined up to 72 h after seeding; however this current had completely disappeared by 18 h after infection with CMV. The delayed rectifier potassium current was detectable in 8% of uninfected HEL cells but, after infection, the proportion of cells expressing this current gradually increased from 20% at 18-24 h post-infection to 100% at 48 h and 72 h. Pharmacological agents known to regulate the activity of ion channels, via cellular secondary messengers, did not alter the frequency at which either current was detected in uninfected and infected cells. Phosphonoformate, an inhibitor of CMV DNA polymerase, caused 95% block of expression of CMV 'late' proteins in infected cells but did not prevent the switching off of the sodium current or the increased expression of the potassium current. The results indicate an association between the expression of CMV 'immediate-early' or 'early' proteins and the down-regulation of the sodium current and up-regulation of the potassium current.

A prospective case-control study of antibodies to coxsackie B virus in idiopathic dilated cardiomyopathy.

OBJECTIVES: This study was conducted to determine the frequency and significance of Coxsackie B virus-specific immunoglobulin-M (IgM) in patients with idiopathic dilated cardiomyopathy and compare them with the frequency in both unmatched and matched control subjects. BACKGROUND: The principal evidence supporting a pathoetiologic role for Coxsackie B viruses in human dilated cardiomyopathy is derived from retrospective serologic studies. These studies have evaluated patients with end-stage disease and have failed to recognize the importance of assessing both matched and unmatched control subjects. METHODS: In this prospective case-control study, we assessed sera for Coxsackie B virus-specific IgM (serotypes B1 to B5) from 114 patients with dilated cardiomyopathy at diagnosis or referral to our center, 94 healthy unmatched control subjects, 41 healthy matched control subjects from the same general practitioner and 32 members of the patients' own households. RESULTS: A higher frequency of positive Coxsackie B virus IgM was observed in patients with dilated cardiomyopathy than in unmatched control subjects (33% vs. 5%; p = 3 x 10(-7)). In patients with dilated cardiomyopathy, the response was monotypic (84%), commonly against serotypes B2 and B5, and was not associated with any clinical or histologic feature. The frequency of positive virus-specific IgM was similar in patients with dilated cardiomyopathy and their 41 matched community control subjects (46% vs. 27%; p = 0.11) and 32 household contacts (37% vs. 28%; p = 0.59). Control subjects who tested positive for virus-specific IgM tended more commonly to be seropositive than did control seronegative subjects (community control subjects 37% vs. 18%, p = 0.32; household contacts 42% vs. 20%; p = 0.36) and had an identical serotypic response in 4 (33%) of 12 cases. CONCLUSIONS: The frequency of Coxsackie B virus IgM was higher in patients with dilated cardiomyopathy than in unmatched control subjects but was similar in patients and control subjects who shared the same environment, indicating local spread of infection. The reason for the association between Coxsackie B virus IgM and dilated cardiomyopathy and its relevance to pathogenesis remain to be established.

Rapid progression of HIV disease in children with cytomegalovirus DNAemia.

OBJECTIVE: In HIV-1-infected children, active cytomegalovirus (CMV) infection can cause severe clinical manifestations and accelerate progression of HIV disease. However, sufficient quantities of blood samples may not be available either for culture or detection of CMV DNA or antigens in white blood cells. The aim of this study was to investigate the diagnostic and prognostic significance of detecting CMV DNA in serum samples from HIV-1-infected children. DESIGN: Sera from 55 children (18 boys), aged 2-130 months (mean, 49.8 months), with perinatal HIV-1 infection and clinical manifestations attributable to CMV infection were tested for CMV DNA by nested polymerase chain reaction and for class-specific CMV antibodies [immunoglobulin (Ig) G, IgA, IgM] by enzyme-linked immunosorbent assay. The children were followed up for 2 days to 59 months (mean, 25.5 months). RESULTS: CMV infection was demonstrated in 43 children (74.5%), 18 of whom (42%) were positive for CMV DNA. During the follow-up, 13 children with CMV infection (30.2%) died, including 11 (84.6%) who were positive for CMV DNAemia just before death. Of these children, seven died soon after hospitalization without antiviral treatment, and four died despite therapy with ganciclovir or foscarnet. Post-mortem CMV inclusions were revealed in seven out of eight children who underwent autopsy. The two other children who died also had progressive CMV disease and received ganciclovir until death. In comparison with CMV-seropositive children without CMV DNAemia, children with CMV DNAemia showed significantly shorter mean survival time (42.5 versus 60 months; P < 0.01), lower final CD4+ T-cell count (218 versus 499 x 10(6)/1; P < 0.01) and higher mortality rate (P < 0.0001). CONCLUSIONS: The detection of CMV DNA in serum is of value for diagnosis of active CMV infection in HIV-1-positive children, and CMV DNAemia is a good prognostic indicator of severe outcome of HIV disease.

Human cytomegalovirus genome sequences in lymph nodes.

Human cytomegalovirus (CMV) is a major cause of morbidity in heart and lung transplant patients, resulting from immunosuppression-mediated reactivation of latent CMV originating either from the transplanted tissue, or the recipient. We showed that out of eight donor/recipient pairs, the lymph nodes (LNs) of three donors and four recipients, all CMV seropositive, harboured CMV DNA at exceeding levels compared with those of matched blood samples, as well as CMV RNA otherwise undetectable in patients' blood. On follow-up, patients positive for CMV DNA and RNA in LNs developed viraemia 4 to 5 weeks earlier than those initially polymerase chain reaction-negative for CMV. Our results indicate that LN are a significant site for sequestration and persistence of CMV and that LN may be important in seeding of CMV-infected cells into the circulation.

Reactivity and assay restriction profiles of monoclonal and polyclonal antibodies to acid phosphatases: a preliminary study.

The development of secure diagnostic immunoassays requires, among others, rigorous characterisation of potential antibody reagents. The reactivity profiles of seven antibodies (six monoclonal [MAb] and one polyclonal [PAb]) with putative specificity for tartrate-resistant acid phosphatase (TRAP) and/or osteoclasts were evaluated in enzyme-linked immunosorbent assay (ELISA) and/or immunocytochemistry. MAbs 2H1, 4E6 and 5Cl demonstrated assay restriction: exhibiting reactivity only in ELISA. The remaining three MAbs (G211D, G312G and V35B) and the PAb 8023 recognised recombinant TRAP (rTRAP) in ELISA and native acid phosphatases in selected tissues and cell lines. The latter were cytochemically assessed for both tartrate-sensitive acid phosphatase (TSAP) and TRAP. V35B showed reactivity against the monocytic leukaemia cell line U937 and guinea pig kidney tissue (both TSAP+ and TRAP+) and ECV304 (TSAP+) cells. Interestingly, the reactivity of MAb G211D co-localised with TRAP activity in the membrane of osteoclasts but also detected cytoplasmic components in U937 cells and human embryonic lung fibroblasts (TRAP+ and TRAP+). G211D exhibited immunoreactivity against placental trophoblasts (positive for total AP). Intriguingly, MAbs 2H1, 4E6, 5Cl and PAb 8023 cross-reacted with potato acid phosphatase in ELISA, suggesting reactivity to conformationally similar epitopes. Thus, some of these reagents could be used in the development of standardised diagnostic immunoassays or as drug-targeting agents for conditions in which the pathological process involves bone resorption, the MAbs G211D, 2H1, 4E6, 5Cl and PAb 8023 being useful in ELISA but not immunocytochemical detection of TRAP.

Sequence analysis of the V3 loop regions of the env genes of Ugandan human immunodeficiency proviruses.

Ugandan strains of human immunodeficiency virus type 1 (HIV-1) were isolated by cocultivation of peripheral blood lymphocytes from infected individuals with cord blood lymphocytes. Sequences from the V3 region of the env gene were amplified by the polymerase chain reaction (PCR) from chromosomal DNA obtained from low passage virus cultures. The PCR products from 13 Ugandan isolates were cloned into a phagemid vector and sequenced. Many isolates contained divergent V3 loop sequences and adjacent regions: diversity was associated with codon deletions or duplications and with nucleotide substitutions, especially G----A transitions. Proviruses from some of the cultures showed extensive diversity within the V3 loop sequences but others were more homogeneous. The V3 loop apices were conserved in 6 of the Ugandan proviruses and these were very similar to the equivalent regions of several Zairean proviruses. The V3 loop apices of African isolates of HIV-1 are divergent from those of North American isolates. The possible biological consequences of this divergence are discussed.

Early detection of cytomegalovirus (CMV) infection in bone marrow transplant patients by reverse transcription-PCR for CMV spliced late gene UL21.5: a two site evaluation.

BACKGROUND: Bone marrow transplant (BMT) patients at risk of developing cytomegalovirus (CMV) pneumonitis are identified routinely by the early detection of virus in blood. For early diagnosis of CMV infection, the RNA-based approach demonstrates advantages when compared with the current CMV antigen and DNA detection methods. OBJECTIVES: We have evaluated our previously developed reverse transcription-polymerase chain reaction (RT-PCR) to a spliced late CMV gene (SLG; J. Virol. Methods 56 (1996), 139) to monitor CMV infection in BMT patients at two clinical sites. The diagnostic value of the SLG RT-PCR was compared with the routine CMV antigen and DNA detection methods. STUDY DESIGN: Weekly blood samples from BMT patients were tested for CMV during the first 3 months post-transplant. The qualitative SLG RT-PCR, semiquantitative DNA PCR, and viral antigen tests were compared. The RNA and DNA PCR results were analysed in terms of their temporal relationship and consistency of CMV detection and compared with CMV infection diagnosed by viral antigen tests. RESULTS: Of the 101 BMT recipients studied, 25 developed CMV antigenemia and/or DNAemia resulting in symptomatic infection in two patients. All CMV PCR-positive patients were either CMV seropositive pretransplant or received marrow from seropositive donor. The highest incidence of CMV infection was seen in seropositive recipients (R+) irrespective of the donor's status. Detection of CMV infection by SLG RNA preceded CMV DNA detection by 0-2 weeks (median 1 week) and CMV antigen detection by 0-8 weeks (median 3 weeks). Once detected, the SLG RNA remained consistently positive before antiviral treatment was commenced. Both the SLG RNA and CMV DNA detection methods had the same clinical sensitivity, specificity, positive and negative predictive values of 100, 94, 80 and 100%, respectively. CONCLUSIONS: The RT-PCR for SLG RNA proved to be the earliest indicator of CMV infection in BMT patients demonstrating a sustained pattern of CMV detection during the 3 months post-transplant period. Although very similar in its diagnostic performance to CMV DNA PCR the SLG RNA RT-PCR does not require quantitation and provides an efficient and ongoing indication of active CMV infection.

Liver function abnormality following treatment with antithymocyte globulin for aplastic anaemia.

We have observed transient elevations of serum alanine transaminase (ALT) levels in patients with aplastic anaemia who have been treated with antithymocyte globulin (ATG). Out of 18 patient episodes analysed retrospectively over a 12 month period, 15 experienced increases in ALT levels with values ranging from 1.2 to 18.5 times the upper limit of normal. In 11 of 15 episodes this was transient with ALT values returning to normal by 30 days, but in two patients this persisted for 6 months, and in a further two, until death at 34 and 145 days from unrelated causes. There was no evidence of acute viral infection or reactivation and no other drug toxicity could be implicated. We conclude that this may represent either a non-specific binding effect of ATG to hepatocytes or infection with an unidentified agent.

Comparison of enzyme-linked immunosorbent assay (ELISA) technique and complement-fixation test for estimation of cytomegalovirus IgG antibody.

The ELISA technique has been found to be reliable for the detection and titration of cytomegalovirus-specific IgG antibody in serum. It is about six times more sensitive than the CF test although some discrepancies were found between the antibody titres determined by the two methods.

Comparison of enzyme-linked immunosorbent assay, radioimmunoassay, complement fixation, anticomplement immunofluorescence and passive haemagglutination techniques for detecting cytomegalovirus IgG antibody.

The radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) techniques were found to be comparable in sensitivity and specificity for detecting cytomegalovirus IgG antibody, and 10 to 100 times more sensitive than complement-fixation (CF), anticomplement immunofluorescence (ACIF) and passive haemagglutination (PHA). In screening tests for antibody, the frequency of false-positive and -negative results was 0.6% for RIA and ELISA, 1.5% for CF, 1.6% for ACIF and 3.6% for PHA. PHA was the least satisfactory test, largely because of technical problems. Cytomegalovirus (CMV) infection is an important cause of congenital brain damage and is also a major complication of both prolonged immunosuppressive therapy, especially in patients with organ transplants, and multi-donor blood transfusions. For serological diagnosis of infection, as well as for screening for antibody in patients and in blood donors, the solid-phase indirect radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) techniques offer distinct improvements in sensitivity over previous methods. Although the principle of both tests, based on the detection of antigen-antibody reactions by means of a labelled anti-antibody, is the same, each possesses its own particular technical advantages and disadvantages, and both require their own expensive equipment for the reading of the results. There is still a lack of data on how they compare in sensitivity and specificity. The present study was undertaken to compare the two methods for the detection of CMV IgG and to evaluate them against the older techniques of complement-fixation (CF), passive haemagglutination (PHA) and anticomplement immunofluorescence (ACIF).

Coxsackie B virus-specific IgM antibody and myocardial infarction.

The ELISA technique was shown to be group-specific for the detection of IgM antibodies against coxsackie B viruses, and probably against a wider range of enteroviruses. No evidence was obtained that recent coxsackie B-virus infection predisposes to myocardial infarction.

Indirect enzyme-linked immunosorbent assay (ELISA) for detection of IgG antibodies against Coxsackie B viruses.

In tests for IgG antibodies against Coxsackie B viruses in man, the enzyme-linked immunosorbent assay (ELISA) was essentially group-specific and, unlike the type-specific neutralisation test, usually failed to detect rises in antibody titre in paired, acute and convalescent, sera. However, in rabbits immunised against Coxsackie B viruses, ELISA demonstrated both group- and type-specific antibody responses. The lack of type-specificity of ELISA in man is probably because repeated infection with enteroviruses--echoviruses and Coxsackie A as well as Coxsackie B--results in masking of the type-specific antibody response by group-specific antibody.

Rapid detection of mumps virus by the polymerase chain reaction.

A procedure for detecting mumps virus in under 48 h was developed using the PCR. The sensitivity of the PCR amplification reaction and of the detection of the PCR product was significantly improved by: (i) enriching for viral template RNAs by overnight culture of the virus in Vero cells and (ii) substitution of polyacrylamide gel analysis for agarose gel electrophoresis. The technique was capable of detecting 1-20 infectious units of virus or an equivalent of 1-10 pg of mumps virus-specific plasmid DNA.

Quantification of cytomegalovirus DNA in blood specimens from bone marrow transplant recipients by the polymerase chain reaction.

A nested PCR system for cytomegalovirus (CMV) DNA in blood specimens from bone marrow transplant recipients is described, in which the biotinylated tritium-labelled product from the second round of PCR is quantified using streptavidin-coated fluorometric Scintillation Proximity Assay (SPA) beads (Amersham, UK). This assay has been compared with a PCR procedure based on limiting-dilution, in which the end-point is determined visually following electrophoresis in agarose gel. The two systems were shown to be equivalent in sensitivity and specificity on testing stored serial blood samples from six CMV antibody-positive allogeneic bone marrow transplant patients who developed viraemia as detected by conventional methods of virus isolation in tissue culture.

The detection of HIV-1 proviral DNA by PCR in clotted blood specimens.

Six hundred and ninety-two specimens, each consisting of a suspension of the residual free cells in samples of clotted blood (but not the clots themselves) from 680 patients at high risk for exposure to human immunodeficiency virus (HIV), were tested by a nested polymerase chain reaction (PCR) procedure which had been optimized to give a sensitivity of detection of one copy of HIV-1 proviral plasmid DNA, and the results were compared to those of testing for antibody to HIV on the same specimens. Fifty-one of the specimens were positive for antibody to HIV and 49 of these were also positive by the PCR; the two samples which gave discordant results were found to be PCR-positive when the test was repeated on DNA extracted from the clots themselves. Two specimens were found to be negative for antibody to HIV but were positive by the PCR (53 positive specimens in all). Direct sequencing of the PCR DNA products confirmed their specificity in all cases and demonstrated that no two patients gave the same predicted amino acid sequence for the V3 loop region. The sequences revealed both European/North American and African motifs at the crown of the V3 loop thus indicating a diversity of HIV strains in the SW Thames Region of South London. The results show that the confirmatory PCR for HIV-1 can be carried out efficiently on the same clotted blood specimens as used for routine HIV serology on patients undergoing diagnostic evaluation.

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.

A polymerase chain reaction to detect a spliced late transcript of human cytomegalovirus in the blood of bone marrow transplant recipients.

A reverse transcription (RT) nested polymerase chain reaction (PCR) procedure is described for detecting RNA to a spliced late gene (SLG) of human cytomegalovirus (CMV), the product of which (175 bp) is easily differentiated in agarose gels from the product when the target is unspliced viral RNA or DNA (258 bp). The SLG-RT-PCR has been compared against a semi-quantitative PCR for CMV DNA in buffy-coat specimens collected weekly after bone marrow transplantation from 3 patients and against the results of culturing these specimens for CMV both by conventional virus isolation, based on the detection of cytopathic effect, and by the early detection of infected cells by staining with virus-specific monoclonal antibodies. The detection of CMV RNA by SLG-RT-PCR correlated well with the detection of infective virus but only when the results of both culture methods were combined, in that neither culture method alone was as sensitive as the SLG-RT-PCR. The presence of SLG RNA in the circulation is of value as a marker of active CMV infection.

Pseudomembranous colitis following clarithromycin therapy.

OBJECTIVE: To describe the association of clarithromycin, used to treat Helicobacter pylori infection and duodenal ulceration, with pseudomembranous colitis in two patients. SETTING: St Mary's Hospital, London, UK. PATIENTS: Two female patients aged 77 and 78 years, admitted with duodenal ulceration and H. pylori infection. INTERVENTION: Clarithromycin (500 mg three times daily) was administered concurrently with omeprazole (40 mg once daily). OUTCOME MEASURES: After an initial improvement in symptoms both patients experienced persistent Clostridium difficile-associated diarrhoea. CONCLUSIONS: High-dose clarithromycin should be used with caution for the treatment of H. pylori infection associated with gastroduodenal ulceration. The drug may induce antibiotic-associated colitis which can lead to morbidity and mortality, particularly in the elderly.

Cytomegalovirus viraemia in HIV infection: association with intercurrent infection.

The aim of this retrospective study was to investigate the clinical significance of cytomegalovirus (CMV) viraemia in HIV-infected subjects (with or without AIDS) who had attended this hospital during a 45 month period. They were reviewed regularly and, when clinically indicated, tested for CMV viraemia. The blood of 105 subjects was cultured for CMV and 34 had at least one episode of CMV viraemia during the review period. The viraemia was present during CMV disease in nine of the 34 positive patients and was the only detectable infection in another two. In the remaining 23 patients, CMV viraemia occurred in association with intercurrent opportunistic infection. Among these 23 patients, the viraemia resolved in 12 after treatment (or natural resolution) of the intercurrent infection and only one of these 12 developed CMV disease (mean review period: 8 months). In another seven patients, CMV viraemia persisted despite treatment (or natural resolution) of the intercurrent infection and four subsequently developed CMV disease (mean review period: 4 months) (P = 0.08, Fisher's exact test). From the remaining four patients, no specimens for CMV culture were obtained after treatment of the intercurrent infection. The CD4 count was higher in the 12 patients in whom there was resolution of the viraemia [mean CD4 60 x 10(6)/l] compared with the seven in whom the viraemia persisted [mean CD4 45 x 10(6)/l]. These findings suggest that in some HIV-positive patients, CMV viraemia was potentiated by intercurrent infection with another micro-organism and that its treatment was sufficient to mitigate the CMV disease.

Threshold integration of bi-amplitude signals.

This study examined the pattern of intensity integration at threshold. The stimuli studied are unique in that they have a compound peak-to-peak amplitude envelope. This waveform was partitioned into two segments, each having a different peak-to-peak magnitude (bi-amplitude). All signals in the bi-amplitude series were the same duration (100 ms). Therefore, threshold differences between these signals are due solely to the integration of intensity in the amplitude dimension. A prediction of the pattern of thresholds, based on the diverted-input hypothesis, suggested that little or no integration would occur when the amplitude difference between segments is greater than a specific magnitude. Our results indicate that there are similarities in the integration process found with variable duration signals and with bi-amplitude signals. We conclude that previous estimates of the minimum intensity level based on temporal integration data underestimates the intensity levels that can contribute to threshold. Our results suggest that there are no apparent constraints on the intensity levels that can be integrated near threshold. The auditory system integrates distributed stimulus intensity in both the time and amplitude dimensions. Temporal integration in the auditory system can be viewed as a signal process, where an enhanced internal representation is given low-level stimuli.