Studies on inactivation of hepatitis B virus with polymerase chain reaction

Polymerase chain reaction-ethidium bromide (PCR-EB) method for detecting hepatitis B virus DNA (HBV-DNA) was established with good specificity and a detection limit of 1 pg HBV-DNA, minimum HBV infection dose in susceptible animal, chimpanzees, could be detected with it. Determination of inactivation of HBV-DNA could be inactivated with active chlorine 1,250 mg/L for 60 minutes, or 2,500 mg/L for 30 minutes, 10 pg HBV-DNA in purified Dane particles could be inactivated by active chlorine 625 mg/L for 10 minutes. Accordingly, use of PCR to evaluate the effects of chlorine disinfectant in inactivating HBV was feasible, and HBV-DNA was a more reliable index for inactivation of HBV than HBsAg.     

Detection of avian leukosis virus subgroup J using the polymerase chain reaction

PURPOSE: To compare neutral, external rotation, and abduction external rotation positions of the glenohumeral joint during magnetic resonance (MR) arthrography in the assessment of the joint capsule, biceps-labral complex, and glenohumeral ligaments. MATERIALS AND METHODS: MR imaging with intraarticular administration of gadopentetate dimeglumine was performed in 10 adult cadaveric glenohumeral joints. Fat-suppressed oblique coronal, oblique sagittal, and axial. T1-weighted spin-echo imaging and axial three-dimensional spoiled gradient-recalled imaging were performed with each shoulder in the neutral, external rotation, and abduction external rotation positions. Shoulders were sectioned in the planes that yielded optimal MR images. Anatomic and MR imaging findings were correlated. RESULTS: The biceps-labral complex was best visualized on oblique coronal and axial images obtained in external rotation. Oblique axial abduction external rotation imaging best delineated the inferior glenohumeral ligament but did not improve assessment of the superior and middle glenohumeral ligaments in comparison with findings in neutral and external rotation. CONCLUSION: Although MR arthrography of the glenohumeral joint clearly delineates the biceps-labral complex and glenohumeral ligaments, external rotation of the shoulder optimizes visualization of the former structures. Abduction external rotation is the best position for evaluation of the inferior glenohumeral ligament and anterior capsular attachment.     

Comparison of human immunodeficiency virus 1 DNA polymerase chain reaction and qualitative and quantitative RNA polymerase chain reaction in human immunodeficiency virus 1-exposed infants

The endothelial lining of the blood-brain barrier tightly controls the distribution of peptide hormones between the central nervous system and the circulation. By using primary cultures of brain microvessel endothelial cells, an in vitro model of the blood-brain barrier, we report here the uptake and transport of the octapeptide angiotensin II by a specific receptor population. With the angiotensin II antagonists losartan (AT1 specific) and PD 123,319 (AT2 specific), we showed that both the uptake and transport of angiotensin II were mediated by the AT1 receptor. Western blot analysis confirmed the existence of the AT1 receptor in our cell-culture model. Rhodamine 123 studies also suggested that both angiotensin II antagonists, but not angiotensin II, were substrates for the P-glycoprotein efflux system, thus restricting the transport of these compounds. These results suggest an AT1 receptor mediates uptake and transport of angiotensin II at the blood-brain barrier and may contribute to the regulation of cerebrovascular levels of the peptide.     

Detection of Shigella and enteroinvasive Escherichia coli using polymerase chain reaction

Two oligonucleotide primers were used in a polymerase chain reaction (PCR) procedure to amplify a region of the invasive-associated locus (ial) of Shigella and enteroinvasive Escherichia coli (EIEC). Detection of the amplified product can be done by agarose gel electrophoresis, which is specific and sensitive enough for routine diagnosis of these two pathogens. PCR is done using DNA extracted directly from faeces. The procedure can be completed in 7 h. These findings demonstrate a novel method for rapid, sensitive, specific, and simple diagnosis of diarrhoea caused by Shigella and EIEC.     

Demonstration of Epstein-Barr viral DNA in paraffin-embedded tissues of Burkitt's lymphoma from Argentina using the polymerase chain reaction and in situ hybridization

Burkitt's lymphoma (BL) is the most frequent non-Hodgkin's lymphoma in children in Argentina. Although epidemiologic studies have linked Epstein-Barr virus (EBV) to more than 90% of African BL cases but to only 10-20% of American and French cases, increased EBV-specific antibody titers were demonstrated in 73% of Argentinian patients with BL. To characterize the relationship between EBV and BL in Argentina, we analyzed paraffin-embedded tissues from 16 cases of BL for the presence of EBV DNA using the polymerase chain reaction (PCR) and in situ hybridization (ISH). PCR analysis showed that only 4 of 16 specimens contained the EBV BamW fragment, and these specimens were all from cases diagnosed in 1984. Results of ISH performed with a specific biotinylated DNA probe against the NotI/PstI fragment of EBV correlated with the PCR findings. EBV sequences were detected with ISH in 70-90% of the tumor cells from the 4 positive cases. These data may suggest an epidemic outbreak of EBV-related BL in 1984 superimposed on sporadic cases of BL, for which EBV may not have been an essential factor. This study also demonstrates the value of using molecular techniques on archival tissue to track epidemiologic trends.     

Detection of Mycoplasma in avian live virus vaccines by polymerase chain reaction

The polymerase chain reaction (PCR) was evaluated to detect mycoplasma contamination of avian live virus vaccines. The specificity of the primers showed that 34 strains belonging to nine species of avian mycoplasma DNA could be detected. The sensitivity of PCR to detect mycoplasma DNA was 10(0.2) colony forming units (cfu) of Mycoplasma synoviae and 10(0.7) cfu of Mycoplasma gallisepticum. When M. synoviae and M. gallisepticum were spiked into several avian live virus vaccines, PCR gave a positive reaction except for the avian pox and the avian encephalomyelitis vaccines which were prepared from organ homogenates. Short-term incubation of avian encephalomyelitis vaccines improved the sensitivity of PCR to detect both M. synoviae and M. gallisepticum. Therefore, PCR, combined with the short-term incubation, were shown to be most effective in detecting mycoplasma contamination in all of avian live virus vaccines.     

Sex identification of turkey embryos using a multiplex polymerase chain reaction

A considerable portion of the W chromosome in Gallinaceous birds consists of tandem repetitive DNA. In the turkey, a 0.4-kb PstI element is repeated about 10,000 times in the female diploid genome but is undetectable as such a unit in males. In this study a multiplex polymerase chain reaction was developed to identify the sex of turkeys based upon the PstI repeat. The technique utilized two pairs of primers, the first pair was designed to amplify a region of the PstI repetitive element, resulting in the production of a 177-bp fragment in females. The other pair was designed to amplify a region of the adenosine triphosphate (ATP) synthase gene, present in both males and females. The simultaneous use of all four primers in the same reaction resulted in the coamplification of a 177-bp and a 250-bp fragment in females and a 250-bp fragment in males. This technique was used to verify the sex of 45 adults of known sex and to identify the sex of 74 embryos from Day 5 to hatch. This procedure is rapid and permits the sexing of many embryos in a short time. The ability to sex early embryos can facilitate studies on avian sex determination.     

Rapid preparation of tissue DNA from paraffin-embedded blocks and analysis by polymerase chain reaction

We have developed a new, easy, and more rapid method for DNA preparation, which avoids contamination. With this method, manual surgical blade scrapings from precisely targeted areas of paraffin block surfaces, without microtome cutting, were used to obtain tissues from 10 different neoplasms. Our results indicate the feasibility of DNA extraction from the scraped paraffin tissue for molecular genetic analysis. We applied this technique successfully to screen for the presence of human papillomavirus using the polymerase chain reaction (PCR) procedure in cases of endocervical, esophageal, and nasopharyngeal carcinomas, and to examine the expression of p53 gene from prostate and gastric adenocarcinomas. We conclude that this procedure is also suitable for purification of PCR products in analysis of the mutation or loss of allelic genes by Bstu I endonuclease digestion.     

High-resolution HLA-DQB1 typing using the polymerase chain reaction and sequence-specific primers

Polymorphism at HLA-DQB1 is known to influence tissue compatibility and disease susceptibility; however, current DQB1 typing methods are unable to distinguish the 32 currently recognized DQB1 alleles. We have developed a 32-reaction PCR-SSP method capable of differentiating all DQB1 alleles that differ in amino acid sequence. This method can resolve all heterozygous combinations of DQB1 alleles, with the exception of several combinations involving alleles not thus far detected in Caucasoid populations.     

Epstein-Barr virus and AIDS-related primary central nervous system lymphoma. Viral detection by immunohistochemistry, RNA in situ hybridization, and polymerase chain reaction

The efficacy of a low dose of mifepristone, 5 mg/day for the first 15 days of the menstrual cycle, followed by medroxy-progesterone acetate (MPA), 10 mg/day for the next 13 days, for inhibiting ovulation was assessed in ten volunteers who were treated for three successive cycles. Hormonal determinations in blood and urine samples, ovarian ultrasonography and an endometrial biopsy taken on day 21-24 of the third treatment cycle were used to monitor the cycles. Ovulation was confirmed in 11 of the 30 treated cycles and, in these 11, the LH peak and follicular rupture occurred during MPA treatment periods. Out of 19 anovulatory cycles, 16 had no increase in progesterone levels and another 3 developed a luteinized unruptured follicle. Progestin administration induced secretory changes in the endometrium, but irregular or delayed development was found. Regular withdrawal bleeding occurred in all subjects. These data indicate that the sequential regimen can suppress ovulation while maintaining regular bleeding but increased efficacy is needed for phase II clinical trials.     

Direct in situ reverse transcriptase polymerase chain reaction for detection of measles virus

Behavioral contexts can evoke a variety of autonomic modes of response, characterized by reciprocal, coactive, or independent changes in the autonomic divisions. The present study investigated the modes of autonomic response to visual illusion and mental arithmetic tasks, by the use of noninvasive measures of sympathetic (pre-ejection period; PEP) and parasympathetic (respiratory sinus arrhythmia; RSA) cardiac control. As previously demonstrated, mental arithmetic was associated with a reciprocal pattern of sympathetic activation and vagal withdrawal. The illusion task, however, yielded a distinct mode of vagal activation in the absence of sympathetic change. Responses within tasks were reliable. In contrast to the general intertask consistency reported for stress tasks that yield similar autonomic modes of response, however, neither PEP nor RSA responses were correlated across the illusion and arithmetic tasks. This may be attributable to the dissimilar modes of autonomic control evoked by these tasks. The distinct modes of autonomic response to arithmetic and illusions emphasize the importance of a bivariate model of autonomic control, and may offer important experimental tools for psychophysiological studies of autonomic control.     

The polymerase chain reaction in pathology

Membrane-fixed CD14 acts as a receptor for the protein-bound endotoxin (LPS) complex and mediates the cellular effects of endotoxin. Soluble CD14 (sCD14) is suggested to neutralize circulating LPS, i.e., acting as an endotoxin antagonist. The aim of this study was to elucidate the release of both sCD14 and endotoxin in traumatized patients, starting from the earliest phase after trauma. A total of 15 patients (O ISS = 19, 9-75) suffering major trauma were enrolled in this prospective study. Blood samples were collected as early as immediately at the site of accident, on hospital admission, and thereafter hourly, then daily. For patients (O ISS = 47) died within 24 h because of their severe injuries. Immediately after the accident as well as during the first 2 h after hospital admission, the mean sCD14 levels of surviving patients did not differ from those of healthy volunteers (n = 53). Thereafter, however, sCD14 increased continuously in the trauma group. The concentrations remained elevated throughout the entire observation period. There was, however, no relation between the sCD14 release and the pattern or the severity of injury. In contrast, endotoxin levels revealed a pattern-specific release. The highest plasma concentrations of LPS were observed in patients suffering from (additional) thoracic injury. On the basis of these results we conclude that the release of sCD14 after trauma does not reflect a strict principle such as action/reaction caused by the appearance of endotoxin immediately after the injury. Soluble CD14 is more likely release by an endotoxin-independent mechanism.     

Detection of neutralizing antibodies against human T-cell leukemia virus type 1 using a cell-free infection system and polymerase chain reaction

We have developed a cell-free infection system to titrate neutralizing antibodies against human T-cell leukemia virus type 1 (HTLV-1) using the polymerase chain reaction (PCR). S+L-CCC (8C) feline kidney or U-251 MG human glioma cells were infected with a cell-free culture supernatant derived from HTLV-1-infected c77 feline cells. DNA was extracted from 8C or U-251 MG cells after incubation for 24 hr and amplified by PCR. The c77 cell supernatant gave discrete bands, whereas those of HTLV-1-positive T cells did not. When the inocula were treated with HTLV-1 antibody-positive human sera or the monoclonal or polyclonal antibody against the peptide 190-199 of HTLV-1 envelope protein gp46, the subsequent formation of HTLV-1 proviral DNA was inhibited. We determined the titers of neutralizing antibodies by densitometrically scanning the intensity of the PCR bands. These titers correlated well with those determined by the plaque assay using a pseudotype of vesicular stomatitis virus bearing the envelope antigens of HTLV-1. At high serum concentrations, many seronegative samples markedly inhibited the plating of the HTLV-1 pseudotype whereas they barely affected results obtained by PCR. Thus, the c77-PCR system can detect neutralizing antibodies against HTLV-1 even at low titers.     

Semiquantitative Epstein-Barr virus (EBV) polymerase chain reaction for the determination of patients at risk for EBV-induced lymphoproliferative disease after stem cell transplantation

Epstein-Barr virus-induced lymphoproliferative disease (EBV-LPD) is a serious and potentially fatal complication after allogeneic stem cell transplantation (SCT). To evaluate levels of EBV DNA in SCT patients, a semiquantitative polymerase chain reaction (PCR) assay was developed. DNA was extracted from peripheral blood leukocytes and diluted, and PCR was performed by using a primer set specific for a well-conserved sequence of the internal repeat 1 region of the EBV genome. Forty-one SCT patients were screened with this method. Thirty-seven patients received allogeneic transplants, of which 18 were T-cell-depleted marrow. Four additional patients received autologous SCT, one of which was T-cell depleted. The mean time of follow-up by EBV PCR was 147 days (range, 47 to 328 days) posttransplant. The range of EBV copies/microg DNA from normal EBV sero-positive donors was 40 to 4,000. Seven patients had >/=40,000 copies of EBV DNA/microg DNA, all of whom were recipients of T-cell-depleted SCT. Five of the seven patients with elevated levels of EBV DNA developed EBV-LPD. Four of these five patients with EBV-LPD had elevated levels of EBV DNA from 1 to 8 weeks before diagnosis. Two patients with EBV-LPD had normal levels of EBV DNA, and two patients with >/=40,000 copies EBV/microg DNA did not develop EBV-LPD. In one patient, clinical resolution of disease correlated with a decrease in EBV DNA and an increase in the level of EBV-specific cytotoxic T-cell precursors. These data indicate that the measurement of EBV viral load with semiquantitative PCR is useful in detecting EBV-LPD in high-risk patients before the onset of clinical symptoms. Because not all patients with elevated levels of EBV DNA develop EBV-LPD, semiquantitative PCR results cannot substitute for clinical, radiographic, and pathological confirmation of this diagnosis.