Down-regulation of integrated Epstein-Barr virus nuclear antigen 1 and 2 genes in a Burkitt lymphoma cell line after somatic cell fusion with autologous EBV-immortalized lymphoblastoid cells.

Epstein Barr virus (EBV) latent gene expression was analyzed in somatic cell hybrids between an EBV-positive Burkitt lymphoma (BL) cell line (BL 60) and an autologous EBV-immortalized lymphoblastoid cell line (LCL, IARC 277). The EBV genomes carried by the parental cell lines differ in sequence and in their physical state. The BL 60 EBV genome is integrated into the host cell genome whereas the LCL IARC 277 carries exclusively episomal EBV molecules. The hybrid cells contain both EBV genomes and display the differentiation phenotype of the parental LCL with regard to growth characteristics and cell-surface antigen expression in vitro and in vivo. While the EBNA 1 and EBNA 2 gene expression of the LCL-derived EBV is maintained in these hybrid cells, the BL-60-derived EBNA 1 and EBNA 2 genes are transcriptionally down-regulated. Mapping of the genomic region surrounding the latent Cp promoter of the BL-60-derived EBV revealed an extensive deletion upstream of the Cp promoter including the enhancer element in the ori P region, the origin of latent viral replication (ori P), the coding sequences for the EBV latent membrane protein (LMP) and the EBV terminal protein (TP), and suggested that one viral-cellular junction sequence is located near the Cp promoter. Integration of EBV into the host cell genome together with the extensive deletion might be causally related with the altered latent gene expression pattern after introduction of a lymphoblastoid host-cell background by somatic cell fusion. Down-regulation of the BL-60-derived EBNA genes could be due to loss of regulatory sequences in the BL-derived EBV necessary for EBNA 1 and EBNA 2 transcription in the lymphoblastoid hybrid cells, but not in the parental BL cells.

The ultrastructure of articular cartilage of the chicken's knee joint.

The articular cartilage and synovial membrane of immature and mature chicken knee joints were studied by light, scanning and transmission microscopy. The findings differed from human articular cartilage and we conclude that the chicken knee joint is not suitable as a model for human joint degeneration.

Sense and antisense transcripts of human papillomavirus type 16 in cervical cancers.

Human papillomavirus type 16 (HPV-16) transcription was analysed in one squamous cervical carcinoma by cDNA cloning and DNA sequencing, and in eight additional squamous cervical carcinomas and 11 precancerous lesions by RNA-RNA in situ hybridization. The nucleotide sequences of the cDNA clones revealed structures of early HPV-16 mRNAs (E6*-E7-E1 E4-E5) in agreement with data reported for other premalignant and malignant tumours. cDNA clones possibly representing viral RNA of antisense orientation were also detected. These RNAs included sequences of the upstream regulatory region, part of the early and the late region of the genome. In three of eight squamous cervical carcinomas examined by in situ hybridization, signals specific for viral antisense RNA were also found. The antisense RNAs had a predominantly nuclear localization. Viral antisense RNA could not be detected in any of 11 HPV-16-positive premalignant lesions. The expression of HPV antisense RNA is likely to be linked to viral integration into the host genome. The possible effects of viral antisense transcription with regard to tumour progression remain to be determined.

Subclinical Epstein-Barr virus infection of both the male and female genital tract--indication for sexual transmission.

Epstein-Barr Virus (EBV) can infect B lymphocytes as well as epithelial cells of the oral cavity. Recently, infection of epithelial cells of the inflamed uterine cervix has been demonstrated, and EBV-DNA has been detected in urethral discharge of men suffering from genital infection. We investigated whether EBV can be found in the genital tract of both sexes independently from inflammatory disease states. Genital specimens of men and women of a sexually transmitted diseases outpatient clinic after excluding sexually transmitted diseases and clinically apparent signs of inflammation were investigated using the polymerase chain reaction to screen for EBV-DNA. In 13 of 47 samples (27.7%) swabbed from the uterine cervix, EBV-DNA could be detected. Similarly, 6 of 45 samples (13.3%) scraped from the sulcus coronarius contained EBV-DNA. Our study shows that the female genital tract and likewise the male genital tract can subclinically harbor EBV. These findings suggest i) that in addition to the oral cavity, the female and the male genital tract may be a reservoir for EBV and ii) that sexual transmission of this virus associated with an epidemiology different from that of oral infection may be possible.

Subchondral vascularisation and osteoarthritis.

A prospective experiment was carried out in 40 rabbits in which the blood supply to the patella was interrupted for defined periods from 2 weeks to 6 months. With prolonged ischaemia, there were clear changes in the articular cartilage which were comparable with those seen in human osteoarthritis.

Subchondral vascularisation and osteoarthritis.

A prospective experiment was carried out in 40 rabbits in which the blood supply to the patella was interrupted for defined periods from 2 weeks to 6 months. With prolonged ischaemia, there were clear changes in the articular cartilage which were comparable with those seen in human osteoarthritis.

Detection of Epstein-Barr virus-DNA in tongue epithelium of human immunodeficiency virus-infected patients.

Oral hairy leukoplakia is a lesion on the lateral part of the tongue that contains replicating Epstein-Barr virus (EBV) and presages progression from human immunodeficiency virus (HIV) infection to AIDS. To clarify the role of EBV in the development of the lesions, we used filter in situ DNA hybridization to determine the prevalence of EBV and of human papillomavirus (HPV) in epithelial cells obtained on swabs from the tongue of HIV-infected patients who had hairy leukoplakia, HIV-infected patients who did not have hairy leukoplakia, and healthy uninfected control persons. In samples collected from the 35 uninfected control persons, EBV DNA could not be detected except at low concentrations in three people. In contrast, all but one of the samples from 11 HIV-infected patients who had hairy leukoplakia contained EBV DNA. Of greatest interest, in 19 of 32 HIV-infected patients who had no signs of hairy leukoplakia, EBV DNA was also detected on the epithelium of the tongue. DNA filter in situ hybridization for the detection of HPV serotypes 6, 11, 16, and 18 in all cases yielded negative results. Statistical analysis showed that the presence of EBV DNA was significantly correlated with the clinical status of the HIV-infected persons, as determined by Walter Reed staging classification, whereas hairy leukoplakia was not. It is concluded that detection of EBV DNA in oral epithelium may be an earlier and more powerful predictor of progression to AIDS than is hairy leukoplakia.

Merkel cell tumor in pregnancy. A case report.

A Merkel cell tumor occurred in a young, pregnant woman. The patient delivered prematurely, and the infant died of prematurity. The patient died within two years of the diagnosis.

Analysis of the HSV-1 strain 17 DNA polymerase gene reveals the expression of four different classes of pol transcripts.

We have investigated the structure and the expression of transcripts of the HSV-1 strain 17 DNA polymerase gene (pol) by various mapping methods including cDNA cloning. The majority of mature pol transcripts is strictly colinear with the pol gene. But additionally, pol cDNAs show a defined heterogeneity in respect to their 5'-terminal regions and can be divided into four classes with characteristic differences; (i) class 1 represents the major transcript (pol-R1) with initiation at HSV-1 positions 62,605-62,610, (ii) class 2 initiates about 70 bp downstream, (iii) class 3 is generated by splicing the short open reading frame (SORF) to a 5'-truncated part of the long open reading frame (LORF) which results in a partially different coding potential, and (iv) class 4 starts 120 bp upstream of the major initiation site in the central part of the origin of replication (oriL). S1 and Exo VII nuclease and RNase protection assays as well as primer extension analyses confirm the classification regarding the genuine structure of pol mRNAs and the differential usage of transcriptional start sites. Furthermore, the transcript classes can be distinguished from each other by their kinetics of appearance/disappearance in the cytoplasm: The first transcription of the pol gene is indicated by the predominant presence of class 2 and class 4 mRNAs at 2 hr postinfection (h.p.i.), followed by an increase of class 1 transcripts up to 4 h.p.i. and a parallel decrease of class 2 mRNAs. These data suggest that expression of the pol gene is finely regulated already at the transcriptional and/or posttranscriptional level prior to the translation of pol mRNAs.

Diagnosis of Epstein-Barr virus infection in hairy leukoplakia by using nucleic acid hybridization and noninvasive techniques.

The presence of Epstein-Barr virus (EBV) DNA in the epithelial cells of oral hairy leukoplakia is the confirming criterion in the diagnosis of this lesion, which occurs mainly in persons infected by the human immunodeficiency virus. Because hairy leukoplakia often presages the development of the acquired immune deficiency syndrome, it is important that suspicious lesions be accurately diagnosed. Commonly, biopsy tissue is removed for detection of EBV DNA by in situ hybridization, but biopsy is contraindicated in some patients. This study evaluated filter and cytospin in situ hybridization, two noninvasive techniques that examine epithelial cells swabbed from the surfaces of the lesions, for their sensitivity in detecting EBV DNA. As compared with tissue in situ hybridization, the filter and cytospin techniques had sensitivities of 100 and 92%, respectively. We conclude that these two noninvasive techniques can provide the clinician with an accurate alternative to biopsy whenever this human immunodeficiency virus-associated lesion is suspected.

[EBV replication and therapy of oral hairy leukoplakia using acyclovir]. / EBV-Replikation und Therapie der oralen Haarleukoplakie mit Acyclovir.

The therapy of choice for oral hairy leucoplakia in HIV-infected patients is treatment with acyclovir. During treatment the replication of Epstein-Barr Virus (EBV) in cells scraped from the epithelium of hairy leucoplakia was investigated using filter-in-situ hybridization. On the 2nd day of treatment a slight and on the 5th day a marked reduction of the replication was observed, and on the 8th day of treatment replication of EBV could hardly be detected. At that time a marked regression of leucoplakia was seen. Within another 7 days the lesions had completely disappeared. These findings demonstrate the relationship between treatment with acyclovir and inhibition of EBV replication in hairy leucoplakia and the relationship between inhibition of EBV replication and remission of hairy leucoplakia.

Early postpartum rooming-in and maternal attachment behaviors in a group of medically indigent primiparas.

Introduction of rooming-in at the study site provided an opportunity to examine the impact of rooming-in on maternal attachment behaviors. Maternal attachment scores for 80 mothers who received rooming-in were compared to 72 mothers who delivered before rooming-in and 35 mothers who requested but did not receive rooming-in. All subjects were medically indigent primiparas with no intrapartum or postpartum complications and term healthy infants. The groups were not significantly different in maternal age, race, or ethnicity. Maternal attachment behaviors were recorded during an infant feeding. Rooming-in mothers had significantly higher maternal attachment scores than both control groups. Rooming-in had an independent effect on maternal attachment after the effects of maternal age, episiotomy or lacerations, epidural anesthesia, infant contact at delivery, and time of feeding observation had been accounted for. Of these prior factors, only maternal age had a significant impact on rooming-in. These results suggest that rooming-in helps primiparas to form early attachments to their babies, and that the impact of rooming-in cannot be explained by the mother's motivation for rooming-in. It is important to provide close contact with the infant during the early postpartum, especially for adolescents who may be at higher than average risk of mothering inadequacies.

Terbutaline (intravenous bolus) for the treatment of acute intrapartum fetal distress.

beta 2-Sympathomimetics have been used in acute intrapartum fetal distress to abolish uterine contractions and thus enable the fetal metabolism to recover before delivery. Because some serious complications were reported when a terbutaline intravenous bolus (0.25 mg) was used as a tocolytic, we assessed its safety and efficacy when used in patients not affected by cardiovascular disease, tachycardia greater than 100 beats/min, thyrotoxicosis, fluid overload, corticoids, atropine, or severe abruptio placentae. No maternal or fetal complications occurred in the 36 patients studied; a well-tolerated tachycardia developed in most patients. Fetal heart rate tracings and pH improved in 32 patients. Thirty-four neonates were delivered in good clinical and metabolic condition. We conclude that terbutaline intravenous bolus 0.25 mg is a safe and efficacious procedure when the proper indications and contraindications are followed.

TPA-inducible Epstein-Barr virus genes in Raji cells and their regulation.

A number of agents including the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) can induce an abortive virus cycle in the EBV nonproducer Burkitt's lymphoma line Raji. We describe the pattern of viral RNAs transcribed in uninduced cells and in cells treated with TPA for 8 hr, as analyzed by Northern blotting. By comparing the patterns of RNAs observed in cells treated with TPA, TPA plus cycloheximide, or cycloheximide alone, we have tested whether any EBV gene in TPA-treated Raji cells would be inducible directly by TPA in the presence of protein synthesis inhibitors, similarly to immediate-early genes induced by superinfection of Raji cells with P3HR-1 virus in the presence of cycloheximide. We demonstrate here that induction of all early EBV genes is dependent on ongoing protein synthesis. The experiments do not provide an answer to whether TPA acts by activating an initial step in the cascade of virus production or whether TPA has a simultaneous pleiotropic effect on the regulation of a large number of viral genes.

A clinical test for diagnosing nuchal cords.

A simple, noninvasive clinical test for detecting nuchal cords late in pregnancy and during labor is based on recording the changes in the fetal heart rate produced by transabdominal manual compression of the fetal neck area. The test was applied in 88 women close to full term and to 67 women in labor. Overall, the sensitivity of the test was 82.3% and specificity, 89.1%. The results were statistically significant in both late pregnancy and labor. A positive test implies an impending risk of cord compression and is an indication for close electronic fetal monitoring, particularly during labor. Routine use of the nuchal cord test can contribute to decreasing perinatal morbidity and mortality by diminishing the impact of cord problems.

Treatment of acute intrapartum fetal distress by beta 2-sympathomimetics.

Patients with a diagnosis of severe intrapartum fetal distress by fetal heart rate and capillary blood pH monitoring received beta 2-sympathomimetics to inhibit uterine contractions (tocolysis) while the obstetric team was preparing to deliver the fetus. Fetal heart rate and acidosis significantly improved after tocolysis; these fetuses were subsequently delivered in very good metabolic and clinical condition. The favorable effect of tocolysis on fetal homeostasis is attributed to the suppression of the ischemic effect of contractions on the placental circulation. The few fetuses having an extremely compromised placental function showed no improvement in heart rate or acidosis with tocolysis and were immediately delivered. Considering the mild side effects observed, the lack of maternal complications, and the remarkable perinatal outcome obtained, we recommend using tocolysis before delivering distressed fetuses.

Genomic localization, sequence analysis, and transcription of the putative human cytomegalovirus DNA polymerase gene.

The human cytomegalovirus (HCMV)-induced DNA polymerase has been well characterized biochemically and functionally, but its genomic location has not yet been assigned. To identify the coding sequence, cross-hybridization with the herpes simplex virus type 1 (HSV-1) polymerase gene was used, as suggested by the close similarity of the herpes group virus-induced DNA polymerases to the HCMV DNA polymerase. A cosmid and plasmid library of the entire HCMV genome was screened with the BamHI Q fragment of HSV-1 at different stringency conditions. One PstI-HincII restriction fragment of 850 base pairs mapping within the EcoRI M fragment of HCMV cross-hybridized at Tm - 25 degrees C. Sequence analysis revealed one open reading frame spanning the entire sequence. The amino acid sequence showed a highly conserved domain of 133 amino acids shared with the HSV and putative Epstein-Barr virus polymerase sequences. This domain maps within the C-terminal part of the HSV polymerase gene, which has been suggested to contain part of the catalytic center of the enzyme. Transcription analysis revealed one 5.4-kilobase early transcript in the sense orientation with respect to the open reading frame identified. This transcript appears to code for the 140-kilodalton HCMV polymerase protein.