Troponin-I positivity in patients referred to Rapid Access Chest Pain Clinic.

BACKGROUND: Rapid Access Chest Pain Clinics (RACPCs) are set up to access patients with new onset chest pain (within the preceding three weeks), of possible cardiac origin. These patients are seen in the clinic within two weeks of referral and the attending physician takes a history, performs a routine clinical examination, and if clinically justified, a treadmill exercise test is performed according to Bruce Protocol. Within the group of patients referred to the RACPC with new onset but otherwise stable angina, there is a potential overlap with patients who in fact may have an evolving acute coronary syndrome, i.e., unstable angina. The aim of this study was to assess the prevalence of Troponin-I positivity as an indicator of acute coronary syndrome. METHODS: This cross-sectional descriptive study included 60 consecutive patients referred to the RACPC with history of recent onset chest pain (within the last three weeks) of possible cardiac origin and positive ETT or confirmed abnormal ischemic ECG at baseline. Troponin-L was measured in these patients. RESULTS: Out of the total 60 patients, 8.33% of the patients referred to RACPC with new onset angina had positive cTnI. CONCLUSION: Point of care test (POCT) for cTnI can help to identify the high risk patient referred to RACPC.

Dengue virus infection induces upregulation of GRP78, which acts to chaperone viral antigen production.

Dengue virus (DENV) pathogenesis is related to the host responses to viral infection within target cells, and therefore, this study assessed intracellular changes in host proteins following DENV infection. Two-dimensional gel electrophoresis and mass spectrometry identified upregulation of the host endoplasmic reticulum (ER) chaperone GRP78 in K562 cells following DENV infection, in the absence of virus-induced cell death. Upregulation of GRP78 in DENV-infected cells was confirmed by immunostaining and confocal microscopy and by Western blot analysis and was also observed in DENV-infected primary monocyte-derived macrophages, a natural target cell type for DENV infection. GRP78 was upregulated in both DENV antigen-positive and -negative cells in the DENV-infected culture, suggesting a bystander effect, with the highest GRP78 levels coincident with high-level DENV antigen production and infectious-virus release. Transfection of target cells to express GRP78 prior to DENV challenge did not affect subsequent DENV infection, but cleavage of GRP78 with the SubAB toxin, during an established DENV infection, yielded a 10- to 100-fold decrease in infectious-virus release, loss of intracellular DENV particles, and a dramatic decrease in intracellular DENV antigen. However, DENV RNA levels were unchanged, indicating normal DENV RNA replication but altered DENV antigen levels in the absence of GRP78. Thus, GRP78 is upregulated by DENV infection and is necessary for DENV antigen production and/or accumulation. This may be a common requirement for viruses such as flaviviruses that depend heavily on the ER for coordinated protein production and processing.

Rapid detection of influenza A virus in clinical samples using an ion channel switch biosensor.

This report describes the fabrication and successful use of the ion channel switch biosensor (ICSB) for rapid point-of-care detection of influenza A in different types of respiratory specimens. Virus culture -- regarded as the "gold standard" -- and an immunochromatographic rapid point-of-care test for influenza A virus were compared with the biosensor. The ICSB rapid test provided an objective readout within 10 min of specimen inoculation into the ICSB chamber wells, without the need for chemical or other pretreatments. Construction of the ICSB with specific antibodies also enables rapid detection and identification of appropriate influenza A subtypes.

Cellular interactions of virion infectivity factor (Vif) as potential therapeutic targets: APOBEC3G and more?

Vif is an HIV accessory protein whose primary function is to negate the action of APOBEC3G, a naturally occurring cellular inhibitor of HIV replication. Vif acts by binding to APOBEC3G, inducing its protein degradation within infected cells and reducing its levels in progeny virions. Interventions that interfere with the Vif-APOBEC3G interaction, raise intracellular or virion associated levels of APOBEC3G, or reduce intracellular levels of Vif, all could hold promise as potential therapeutic approaches aimed at enhancing the cells innate antiviral activity. Levels of APOBEC3G might be increased or Vif levels decreased, by strategies targeting protein synthesis, protein degradation or cellular localisation and function, and properties of APOBEC3G and Vif relevant to these strategies are discussed. Recent data have suggested that Vif may have other mechanisms of action apart from the above activities against APOBEC3G, including effects against other anti-viral mechanisms independent of APOBEC3G cytidine deaminase activity. In addition to interaction with APOBEC3G, Vif may have other accessory functions, which are discussed in relation to potential therapies that may affect multiple stages of the HIV life cycle. Future development of strategies that combine enhancement of APBOEC3G functional with inhibition of multiple Vif functions may become useful tools for HIV therapy.

Vif-deficient HIV reverse transcription complexes (RTCs) are subject to structural changes and mutation of RTC-associated reverse transcription products.

Reverse transcription (RTn) in HIV-infected cells occurs in a nucleoprotein complex termed the reverse transcription complex (RTC). RTCs containing RT activity and integrase (IN) were shown to be heterogeneous in size and density on sucrose velocity and equilibrium gradients. WT and Vif-deficient (Deltavif) RTCs produced by infection with virus from permissive cells displayed similar sedimentation characteristics, while RTCs from Deltavif virus produced in non-permissive cells demonstrated a reduction in the major RTC form and more of the RTn products in rapidly sedimenting structures. APOBEC3G derived from virions did not co-sediment with RTCs, but RTCs from Deltavif infections showed elevated levels of mutations in RTn products, consistent with APOBEC3G and other mutational mechanisms. The most mutated transcripts were present within rapidly sedimenting RTCs. Thus, virus without functional vif, produced from non-permissive cells, forms abnormal RTCs that contain increased mutation of RTC-associated RTn products in newly infected target cells.

Disease progression and adverse events in patients listed for elective percutaneous coronary intervention.

OBJECTIVE: To record disease progression and the timing of adverse events in patients on a waiting list for elective percutaneous coronary intervention (PCI). DESIGN: Observational prospective study. SETTINGS: A UK tertiary cardiothoracic centre, at a time when waiting lists for PCI were up to 18 months. PATIENTS: 145 patients (116 men, median age 59.5 years) placed on an elective waiting list for PCI between October 1998 and September 1999. MAIN OUTCOME MEASURES: Adverse events recorded were death, myocardial infarction, need for urgent hospital admission because of unstable angina, and need for emergency revascularisation while waiting for PCI. RESULTS: During a median follow up of 10 months (range 1-18 months), nine (6.2%) patients experienced an adverse event. Eight (5.52%) patients were admitted with unstable angina as emergencies. One was admitted with a myocardial infarction. Twenty nine (20.0%) patients had significant disease progression at the time of the repeat angiogram before PCI. In 10 (7%), disease had progressed so that PCI was no longer feasible and patients were referred for coronary artery bypass graft. Sixteen (11%) were removed from the PCI waiting list because of almost complete resolution of their anginal symptoms. CONCLUSION: Adverse coronary events and clinically significant disease progression occur commonly in patients waiting for PCI. Despite the presence of severe coronary lesions, myocardial infarction was rare and no patients died while on the waiting list. Resolution of anginal symptoms was also comparatively common. The pathophysiology of disease progression frequently necessitates a change in the treatment of patients waiting for PCI.

A duck hepatitis B virus strain with a knockout mutation in the putative X ORF shows similar infectivity and in vivo growth characteristics to wild-type virus.

Hepadnaviruses including human hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) express X proteins, HBx and DHBx, respectively. Both HBx and DHBx are transcriptional activators and modulate cellular signaling in in vitro assays. To test whether the DHBx protein plays a role in virus infection, we compared the in vivo infectivity and growth characteristics of a DHBV3 strain with a stop codon in the X-like ORF (DHBV3-X-K.O.) to those of the wild-type DHBV3 strain. Here we report that the two strains showed no significant difference in (i). their ability to induce infection that resulted in stable viraemia measured by serum surface antigen (DHBsAg) and DHBV DNA, and detection of viral proteins and replicative DNA intermediates in the liver; (ii). the rate of spread of infection in liver and extrahepatic sites after low-dose virus inoculation; and (iii). the ability to produce transient or persistent infection under balanced age/dose conditions designed to detect small differences between the strains. Thus, none of the infection parameters assayed were detectably affected by the X-ORF knockout mutation, raising the question whether DHBx expression plays a physiological role during in vivo infection with wild-type DHBV.

Kinetics of human immunodeficiency virus type 1 (HIV) DNA integration in acutely infected cells as determined using a novel assay for detection of integrated HIV DNA.

We have developed a novel linker-primer PCR assay for the detection and quantification of integrated human immunodeficiency virus type 1 (HIV) DNA. This assay reproducibly allowed the detection of 10 copies of integrated HIV DNA, in a background of 2 x 10(5) cell equivalents of human chromosomal DNA, without amplifying extrachromosomal HIV DNA. We have used this assay and a near-synchronous one-step T-cell infection model to investigate the kinetics of viral DNA accumulation following HIV infection. We report here that integrated HIV DNA started accumulating 1 h after the first appearance of extrachromosomal viral DNA and accounted for approximately 10% of the total HIV DNA synthesized in the first round of viral replication. These results highlight the efficient nature of integrase-mediated HIV integration in infected T cells.

Specific inhibition of human immunodeficiency virus type 1 (HIV-1) integration in cell culture: putative inhibitors of HIV-1 integrase.

To study the effect of potential human immunodeficiency virus type 1 (HIV-1) integrase inhibitors during virus replication in cell culture, we used a modified nested Alu-PCR assay to quantify integrated HIV DNA in combination with the quantitative analysis of extrachromosomal HIV DNA. The two diketo acid integrase inhibitors (L-708,906 and L-731,988) blocked the accumulation of integrated HIV-1 DNA in T cells following infection but did not alter levels of newly synthesized extrachromosomal HIV DNA. In contrast, we demonstrated that L17 (a member of the bisaroyl hydrazine family of integrase inhibitors) and AR177 (an oligonucleotide inhibitor) blocked the HIV replication cycle at, or prior to, reverse transcription, although both drugs inhibited integrase activity in cell-free assays. Quercetin dihydrate (a flavone) was shown to not have any antiviral activity in our system despite reported anti-integration properties in cell-free assays. This refined Alu-PCR assay for HIV provirus is a useful tool for screening anti-integration compounds identified in biochemical assays for their ability to inhibit the accumulation of integrated HIV DNA in cell culture, and it may be useful for studying the effects of these inhibitors in clinical trials.

Rapid and efficient cell-to-cell transmission of human immunodeficiency virus infection from monocyte-derived macrophages to peripheral blood lymphocytes.

Macrophages are considered of central importance in cell-to-cell transmission of human immunodeficiency virus (HIV) infection in vivo. In this report, we describe a novel cell-to-cell transmission model using HIV-infected monocyte-derived macrophages (MDMs) as donor cells and peripheral blood lymphocytes (PBLs) as recipients. Virus was transmitted during a 2-h coincubation period from intracellular or tightly cell-associated viral stores in adherent infected MDMs to nonadherent CD3(+) PBLs. Transmission required cell contact, but syncytia formation was not observed. HIV cell-to-cell transmission occurred in both allogeneic and autologous systems, and replication was higher in phytohemagglutinin (PHA)-stimulated than unstimulated recipient PBLs. In contrast, transmission of infection by cell-free virus was barely detectable without PHA stimulation of recipients, suggesting the cell-cell interaction may have provided stimuli to recipient cells in the cell-to-cell system. Viral DNA levels increased 5-24 h postmixing, and this increase was inhibited by pretreatment of cells with the reverse transcription inhibitor azidothymidine, indicating de novo reverse transcription was involved. Cell-to-cell transmission was more efficient than infection with cell-free virus released from donor MDMs, or 0.1 TCID(50)/cell cell-free viral challenge. This model provides a system to further investigate the mechanisms and characteristics of HIV cell-to-cell transmission between relevant primary cells that may be analogous to this important mode of virus spread in vivo.

Kinetics of early molecular events in duck hepatitis B virus replication in primary duck hepatocytes.

This paper describes the use of one-step growth conditions to study the kinetics of duck hepatitis B virus (DHBV) replication in primary duck hepatocytes. Synchronized infection was achieved using partially purified DHBV virions at an m.o.i. of 640 DHBV DNA-containing virions per cell, and these conditions were shown to produce a single cycle of infection. In this model, input purified DHBV DNA was rapidly internalized by cells at > or = 0.5 h, and localized to the nucleus by 4 h, but both covalently closed circular (CCC) DNA and single-stranded DNA were not detected until 48 h postinoculation (p.i.), suggesting that there was a > or = 40 h delay between DHBV localization to the nucleus and formation of CCC DNA. In contrast, CCC DNA can be first detected in hepatocytes at 6 h p.i. in in vivo infection of ducks with the same DHBV strain. In an analysis of the nuclear transport of the DHBV genome, release of nuclear viral DNA from a particulate form to a soluble nucleoplasmic form was only 50% complete by 48 h p.i. However, this process occurred simultaneously with genome uncoating since all soluble nucleoplasmic DHBV DNA was free of nucleocapsid material; this suggests that nucleocapsid disassembly and genome uncoating may occur at the nuclear membrane and not within the nucleus. Quantitative analysis demonstrated inefficiency in a number of steps including virus uptake and internalization, translocation of nucleocapsid across the nuclear membrane and antigen expression from intranuclear viral DNA.

Diagnostic cardiac catheterisation in a hospital without on-site cardiac surgery.

OBJECTIVE: To assess the feasibility, safety, and clinical impact of diagnostic cardiac catheterisation in a multipurpose laboratory in a district general hospital without cardiac surgery. METHODS: A prospective audit of the first 2000 consecutive cases between September 1992 and March 1997. Unstable patients were referred to a surgical centre for investigation, in line with subsequently published British Cardiac Society (BCS) guidelines, but all other patients requiring cardiac catheterisation were investigated locally and are included in this report. The function of the laboratory was also compatible with the BCS guidelines regarding staffing, operators, equipment, number of cases, and locally available vascular surgery. RESULTS: Of the 2000 cases, 1988 studies were completed (99%), 1985 (99%) included coronary angiography, and 1798 (90%) were performed as day cases. Left main stem disease was present in 157 (8%), three vessel disease in 683 (34%), two vessel disease in 387 (19%), single vessel disease in 424 (21%), and normal coronary arteries in 494 (25%). Of the latter, 284 (14% of the total) had another cardiac diagnosis for which they were investigated (for example, valvar heart disease). Referral for cardiac intervention following catheterisation was made in 1172 of the 2000 cases (intervention rate 59%; catheter:intervention ratio 1. 7:1). The interventions performed were coronary artery bypass grafting (CABG) in 736 of the 1172 cases (63%), other types of cardiac surgery in 122 (10%), combined CABG and other cardiac surgery in 71 (6%), and percutaneous transluminal coronary angioplasty in 243 (21%). There were two catheter related deaths (0. 1%), both of which occurred within 24 hours of the procedure, and a further nine major cardiovascular complications with residual morbidity (0.45%). These were myocardial infarction in two (0.1%), cerebrovascular events in two (0.1%), and surgical vascular complications in five (0.25%). In addition, there were eight successfully treated, life threatening arrhythmias (0.4%). CONCLUSIONS: Diagnostic cardiac catheterisation can be performed safely and successfully in a local hospital. When BCS guidelines are followed, the mortality is similar to published pooled data from regional centres (0.1% v 0.12%). The high intervention rate indicates a persistent unmet demand in the districts, which will continue to affect surgical and interventional services.

Isolated clinic hypertension is not an innocent phenomenon: effect on the carotid artery structure.

This study examines the common carotid intimal-medial wall thickness (CCA-IMT) in untreated patients with elevated clinic blood pressure (BP) but normal ambulatory BP (isolated clinic hypertension, n = 22), in comparison with a group with elevated clinic and ambulatory BP (hypertensives, n = 41) and a group with normal clinic and ambulatory BP (normotensives, n = 17) readings. The three groups did not differ in age, male/female ratio, lipid profile, glucose tolerance test, or smoking habits. No difference existed in CCA-IMT values between the groups with hypertension (0.67 +/- 0.18 mm) and isolated clinic hypertension (0.68 +/- 0.14 mm), but the values in these two groups were significantly higher (one-way ANOVA; F = 8.09, P < .001) than in the group of normotensives (0.50 +/- 0.09 mm). The CCA-IMT did not correlate with clinic systolic or diastolic BP readings or with BP derivatives of 24-h ambulatory monitoring. Mean 24-h BP in the isolated clinic hypertensives did not differ from that in the normotensives, whereas both were lower than in the hypertensives. We conclude that changes in the CCA-IMT occuring in subjects with isolated clinic hypertension are equal to the changes in sustained hypertension, indicating that isolated clinic hypertension may not be a benign condition.

Interference between effector RNAs expressed from conventional dual-function anti-HIV retroviral vectors can be circumvented using dual-effector-cassette retroviral vectors.

Coexpression of different effector molecules from a single vector (a dual-function vector) may provide enhanced efficacy. Thus far most of the reported anti-HIV dual-function vectors express different effector RNAs as a chimeric molecule. In our study involving retroviral vectors coexpressing a U5 ribozyme and either an anti-tat or anti-rev antisense RNA, chimeric vectors exhibit poor potency in several important functional aspects, including inhibition of HIV replication, protection against cytopathic effects, and suppression of target gene function. Surprisingly, such a poor efficacy of chimeric vector function was not associated with a lower level of effector RNA expression. These results indicate that expression of two effector RNAs as a chimeric molecule can lead to interference, reducing their global biological effects. More importantly, we have demonstrated that such interference can be avoided by coexpressing these effector RNAs as separate molecules through a new dual-function vector, called a dual-effector cassette (Dec) vector, developed in this study. We also define some of the design alterations that might affect the efficacy of the Dec vector and demonstrate that forward-designed Dec vectors are more efficacious than reverse-designed Dec vectors, which express a lower level of effector RNA owing to the instability of the 5' effector cassettes in the provirus. We believe that the principle of Dec vector design may also be applicable for the coexpression of other therapeutic RNA effectors in many gene therapy applications.

Further characterization of HIV RNA synthesis early after cell-to-cell transmission infection.

Using a one-step model for cell-to-cell transmission of HIV infection we have identified two distinct phases of HIV RNA synthesis. The first phase (4 h-12 h p.i.) was marked by an increase in only the full-length 9 kb RNA, while the second phase (24 h p.i. onwards) comprised a significant increase in the levels of all three species of viral RNA. We now report that while the continual presence of actinomycin D at 50 micrograms/ml abolished all detectable viral nucleic acid synthesis when virus donor H3B cells were pre-treated with 50 micrograms/ml of actinomycin D (AmD), washed free of unbound drug (a procedure which inhibited > 99% of total cellular RNA transcription) then mixed with untreated recipient Hut 78 cells, normal amounts of full length linear unintegrated viral DNA were produced and the first phase of viral RNA transcription was unaffected. Similarly, when both the virus donor cells and recipient cells were arrested in the late G1 phase of the cell cycle by aphidicolin and then mixed, linear unintegrated viral DNA was the major viral DNA species produced. The appearance of circular viral DNA and progeny virus was inhibited, but the first phase of induced viral RNA synthesis was unaffected. When AZT was added at 2 h or 4 h after cell-cell mixing, the level of 9 kb RNA detected was significantly lower, corresponding to reduction in the level of viral DNA. These and previous results indicate that the template for the first phase of viral RNA synthesis was likely to be newly synthesized, linear unintegrated viral DNA and not pre-existing proviral DNA present in the donor cells. Taken together, our results suggest that there exists a yet to be fully characterized pathway of concurrent viral DNA and RNA synthesis early after cell to cell transmission of HIV infection.

Characterization of age- and dose-related outcomes of duck hepatitis B virus infection.

Experimental inoculation of naive ducks with duck hepatitis B virus (DHBV) can lead to one of three outcomes, namely, persistent viremia, transient infection with or without viremia, or no evidence of infection. The ability of individual ducks to resolve DHBV infection was found to be linked to the age of the duck at the time of inoculation and the dose of inoculated virus. (1) In recently hatched ducks inoculated intravenously (i.v.) with 4 x 10(4) DHBV DNA genomes, a switch from persistent viremia to transient antibody appearance was seen at an age of inoculation between 7 and 14 days. A 25-fold increase in the dose of virus (1 x 10(6) DHBV genomes) delayed this switch by 7 days. (2) When 4-month-old ducks were inoculated i.v. with different doses of virus, only those receiving the highest dose (2 x 10(11) DHBV genomes) showed viremia and extensive viral replication and histological changes in the liver; 2/3 ducks in this group had a transient infection, while the third duck had viral replication and histological changes in the liver that were still present at day 120 postinoculation (p.i.). In all ducks receiving lower doses (1 x 10(3), 1 x 10(6), 1 x 10(9) DHBV genomes) antibodies to viral surface and core antigens developed without detectable viral replication in the liver on days 6, 9, or 12 p.i. (3) When 10- to 16-month-old ducks were inoculated i.v. with 2 x 10(11) DHBV genomes, all showed extensive viral replication in hepatocytes and mild to moderate histological changes in the liver on days 4 or 6 p.i. In 4/5 ducks viremia was not detected, anti-surface antibodies were first detected on day 8 p.i., and viral DNA and antigen were cleared from the liver by days 35-47 p.i. The remaining duck became viremic with persistence of virus in the liver until at least day 46 p.i. The findings of the study are consistent with a model for noncytopathic viruses (R. M. Zinkernagel (1996) Science 271, 173-178).

Evaluation of an ultrasonically guided venepuncture technique for the placement of permanent pacing electrodes.

We have evaluated a method of puncturing the subclavian vein in its extrathoracic portion using an ultrasound guidance system. Seventy consecutive patients requiring permanent pacemakers were included in the study. The method was successful in 56 (80%) cases (23 dual chamber systems) and unsuitable or unsuccessful in 14 (20%) cases (2 dual chamber systems). The time taken to achieve a successful cannulation of the vein was similar to that taken with conventional subclavian venepuncture (average time taken for each venepuncture was 31 seconds, range 5-130 seconds). There was a significant "learning curve" in that nearly all of the unsuccessful cases were in the first half of the series. There were no major complications. Computerized Tomography (CT) confirms that the point of entry into the subclavian vein using this technique lies outside the thoracic cavity, thereby minimizing the risk of pneumothorax. This approach to the subclavian vein is an easy technique to learn, with few immediate complications and there may be less chance of lead fracture due to subclavian crush in the longer term.