High levels of hepatitis B virus DNA in body fluids from chronic carriers.

Chronic infection with hepatitis B virus (HBV) is a major global health problem. Transmission is mainly blood-borne, although the route of infection during horizontal transmission in childhood is unclear. Nosocomial outbreaks of HBV have been widely reported, but have mainly focused on blood-borne transmission. There is limited knowledge of the viral load levels in other body fluids. In the present study, chronic HBV carriers were tested for the presence of HBV DNA in serum, saliva, nasopharyngeal fluid, urine and tears by means of qualitative and quantitative polymerase chain reaction (PCR) methods. Twenty-five patients who were positive for HBV DNA with both PCRs were included. Low titres in real-time PCR corresponded with weak bands in the qualitative assay. HBV DNA was found in two urine samples, 10 saliva samples, five nasopharyngeal swabs and in tear fluid from four patients. One highly viraemic HBeAg-positive carrier with serum HBV DNA levels of 7 x 10(9) genome copies had high copy numbers detected in both saliva and nasopharyngeal fluid. These results demonstrate that highly viraemic HBV carriers may have high titres of HBV DNA in other body fluids. This has particular importance for infection control programmes and regulations, underlining the importance of aiming towards regular HBV DNA testing and thus infectivity assessment of chronic carriers in order to prevent transmission.

Peptide presentation by bacteriophage P4.

This article focuses on bacteriophage P4 as a potential peptide display phage by exploring the possibility of using the P4 capsid decoration component, Psu, as a peptide carrier protein. Psu is non-essential for P4 growth but it enhances the stability of the P4 capsid by binding to its exterior. We have constructed a unique SacI cloning site in the beginning of the psu gene. This site changes the third amino acid of Psu from Ser to Leu. This substitution does not destroy the binding of Psu to the P4 capsid. A synthetic oligonucleotide encoding a 10-amino acid peptide whose sequence is part of the human p62c-myc protein, has been inserted into the SacI site. The Psuc-myc shows full capsid binding activity and reacts with monoclonal antibodies directed against the c-myc peptide. These results pave the way for the further development of a peptide display system based on bacteriophage P4.

The capsid size-determining protein Sid forms an external scaffold on phage P4 procapsids.

Although the phages P2 and P4 build their capsids from the same precursor, the product of the P2 N gene, the two capsids differ in size: P2 builds a 60 nm, T = 7 capsid from 420 subunits, whereas P4 makes a 45 nm, T = 4 capsid from 240 subunits. This difference leads to substantial changes in shell geometry and subunit interactions. Previous results have demonstrated that the P4 sid gene is responsible for the assembly of P4-sized shells. We have used cryo-electron microscopy and image reconstruction to determine the structure of a putative assembly intermediate of P4 capsids, produced in vivo from cloned genes. We demonstrate that Sid forms a P4-specific scaffold with icosahedral symmetry on the outside of the procapsid-like particles. The Sid molecules (60 or 120 copies) form lofty arches that interact with the gpN hexamers on the icosahedral 2-fold axes, and connect as trimers over the 3-fold axes, forming a continuous dodecahedrally shaped outer cage. The gpN shell inside the Sid cage is approximately 40 nm wide, consistent with the previously suggested maturational expansion. The main difference with respect to the mature P4 capsids is found in the hexamers, which appear strongly elongated and more protruding than in the mature shell. These and previous results are discussed in the light of a model for regulation of capsid size.

Directed mutagenesis of the bacteriophage P2 ogr gene defines an essential function.

The ogr gene of bacteriophage P2 codes for a basic protein of 72 amino acids which is thought to be essential for activation of P2 late gene transcription. However, conditionally lethal mutations in the ogr gene have never been isolated. We have constructed a P2 ogr deletion mutant by in vitro techniques. This deletion phage, P2-del15, grows in a host which provides the ogr gene product in trans from a plasmid but fails to grow in hosts lacking the ogr plasmid. This demonstrates that the ogr gene is essential for P2 lytic growth. The deletion in P2del15 has removed about half of the carboxy-terminal part of the ogr gene. The transcript from this deletion mutant can be distinguished from the wild-type transcript by S1 nuclease protection. The analysis of such transcripts suggests that the ogr gene product may negatively regulate its own transcription.

Photoimmunodetection: a nonradioactive labeling and detection method for DNA.

We describe a simple detection system for DNA based on antibody detection of UV-induced photoproducts. It includes a convenient and inexpensive labeling procedure, which is completed in 5-10 min. The only equipment required is a UV source such as an ordinary transilluminator or a DNA crosslinker. Using a monoclonal antibody specific for thymine dimers, coupled to horseradish peroxidase, we are able to detect subpicogram amounts of UV-irradiated DNA directly, and approximately 10 pg homologues DNA indirectly by hybridization with an irradiated probe.

On the prognostic value of systemic methotrexate clearance in childhood acute lymphocytic leukemia.

The prognostic value of systemic methotrexate clearance (ClMTX) during high-dose therapy was evaluated in a cohort of 42 children with acute lymphocytic leukemia (ALL). As part of an extensive chemotherapy protocol, they had received a total of 293 methotrexate (MTX) infusions in the 6-8 g/m2 dose range. At the termination of the study, when they had all been followed up for 3.5 years or more, 26 of these patients were still in continuous complete remission, whereas 16 had suffered relapse. The intrapatient variability in ClMTX during the eight courses was up to six-fold. In 67% of the patients, the maximum level of ClMTX reached at least twice the minimum value. The coefficients of variation for the intra- and interindividual variability in ClMTX were 9-57% and 26-41%, respectively. The cumulative probability of relapse, estimated by the Kaplan-Meier procedure, was increased for patients with a high ClMTX during the initial treatment course, but the difference was not significant on a 5% level. There was no significant relationship between high individual median ClMTX and subsequent relapse of ALL. However, ClMTX during the initial infusion, the time-dependent mean for ClMTX, and the individual patient's median ClMTX, were significant predictors for event-free survival in a Cox proportional hazards regression analysis. The present study demonstrates gross pharmacokinetic variability and unpredictable values of ClMTX in subsequent courses after standardized administration of MTX to paediatric patients with ALL. In spite of the association between ClMTX and prognosis shown by some of the analyses, estimates of ClMTX rates may not necessarily be related to disease outcome in a way that can be exploited to the benefit of the individual patient.

Variability in methotrexate serum and cerebrospinal fluid pharmacokinetics in children with acute lymphocytic leukemia: relation to assay methodology and physiological variables.

Methotrexate (MTX) steady state concentrations were evaluated in 42 children who had received high-dose infusions (6-8 g/m2) for acute lymphocytic leukemia. Concentrations in serum and cerebrospinal fluid (CSF) measured by immunoassay were found to be highly variable. Reanalysis by a reference high-pressure liquid chromatography method ruled out analytical factors as a source of this variability. The correlation coefficient between the analytical methods was 0.77 for the serum data and 0.88 for the CSF data. The variability of serum and CSF concentrations was higher in younger patients (serum; P = 0.05 and CSF; P = 0.18), and the CSF concentration decreased with decreasing age and in later courses. Body surface area, body mass index, weight, and gender were not significantly related to MTX variability. We conclude that the pronounced pharmacokinetic variability seen during MTX infusions remains largely unexplained.

Detection of genital papillomavirus types by polymerase chain reaction using common primers.

We describe the detection of eight genital human papillomavirus (HPV) types, including HPV16 and HPV18, by PCR amplification of a 323 base-pair region of the genome within the L1 open reading frame (ORF). The primer sequences are: TGYAAATATCCWGATTWTWT and GTATCWACMACAGTAACAAA. The method will detect purified HPV16 DNA down to a concentration of as little as a single molecule in 100 microliters. The method is also applicable to purified DNA and crude lysates from tumour biopsies. Typing of the PCR product can be achieved with specific oligonucleotide probes.

Human papillomavirus infection in Norwegian women with cervical cancer.

The objective of the present study was to determine the prevalence of human papillomavirus (HPV) infections in Norwegian women with cervical cancer. We used the polymerase chain reaction (PCR) and Southern blot techniques to assess the prevalence of HPV in cervical biopsies of 133 women admitted to the Norwegian Radium Hospital for treatment of cervical cancer. At the time of sampling (from February 1988 to April 1989) about 85% of Norwegian women with cervical cancer were treated at the Norwegian Radium Hospital. HPV was found in biopsies of 91 (68%) of women with cancer; 70 (53%) biopsies contained HPV type 16, 19 (14%) HPV type 18, 4 (3%) HPV type 33, 2 (1.5%) HPV type 11, and 3 (2%) HPV DNA of unknown type (HPVX). Five percent of biopsies were doubly infected, chiefly with HPV 16 + 18. We found a significant association between HPV 18 and low age, poorly differentiated tumors and adenocarcinomas. Our results show that there is an association between HPV types 16 and 18 and cervical cancer also in a Norwegian setting. PCR was more sensitive than Southern blotting for detection of HPV. Thirty-six (27.5%) of cancer biopsies were positive by PCR but negative by Southern blotting, as against 49 (73.5%) positive by both methods; we also encountered 4 samples positive by Southern blotting and negative by PCR. In 23/53 cancer biopsies positive by Southern blotting we found evidence for integrated or rearranged HPV genomes.

The prevalence of circulating anti-tubular basement membrane-antibody in renal diseases, and clinical observations.

Anti-tubular basement membrane antibodies were determined by ELISA in 217 patients with different renal diseases. The assay for antibodies in serum was based on a 58 kD bovine tubular basement membrane antigen. Sera were studied from 69 patients with different forms of interstitial nephritis; 15 patients (10 women, 5 men) had anti-tubular basement membrane titers above the normal (compared with a reference group of healthy blood donors). Three patients are presented in greater detail. Thirty-four patients with pyelonephritis (confirmed by intravenous urogram) were investigated; one serum was positive. Sera from 114 patients with renal glomerular and/or vascular disease were studied; 12 had positive titers for tubular basement membrane and glomerular basement membrane or other kidney disease antibodies. This study supports the opinion that damage in the renal medulla can be caused by an autoimmune process. Circulating anti-TBM antibodies may be of value in the investigation of patients with tubulo-interstitial diseases but the cause and prognosis of this condition is, however, not known.

The reaction between epichlorohydrin and polysaccharides: Part 1. Syntheses of some model substances with non-cyclic substituents.

Five derivatives of methyl alpha-D-glucopyranoside, in which the substituents form noncyclic structures, have been prepared as model substances for possible structural elements formed on reaction of polysaccharides with epichlorohydrin. The substances were converted into the permethylated alditol-1-d derivatives and characterised by CIMS and EIMS.

The reaction between epichlorohydrin and polysaccharides: Part 2, Synthesis of some model substances, with cyclic substituents.

Eight derivatives of methyl alpha-D-glucopyranoside, in which the substituents are part of cyclic structures, have been prepared as model substances for possible structural elements formed on reaction of polysaccharides with epichlorohydrin. The substances were converted into the permethylated alditol-1-d acetates and characterised by CIMS and EIMS.

Structural studies of the capsular polysaccharide from Streptococcus pneumoniae type 1.

The capsular polysaccharide from Streptococcus pneumoniae type 1 is composed of D-galactopyranosyluronic acid residues and 2-acetamido-4-amino-2,4,6-trideoxy-D-galactopyranosyl residues. The latter sugar, previously unknown in Nature, was not isolated but was identified from the products obtained on deamination of the polymer. Using n.m.r. spectroscopy, methylation analysis, and Smith degradation as the principal methods of structural investigation, it is concluded that the polysaccharide is composed of trisaccharide repeating-units having the structure: leads to 3)-alpha-Sugp-(1 leads to 4)-alpha-D-GalpA-(1 leads to 3)-alpha-D-GalpA-(1 leads to, in which Sug denotes the new sugar.

Planning a health education programme for the elderly visually impaired person--a focus group study.

Focus group methodology was used with the aim of learning how persons with the diagnosis of age-related macular degeneration perceived and described their disease, and how the disease had changed their activities of daily living (ADL). This information is seen as critical in designing a health education programme. The focus group participants described problems in performing ADL. Factors contributing to the ADL problems were categorized as functional limitations in, and feelings about performing, ADL. The participants used a number of ADL strategies to adapt to new situations. The participants expressed uncertainty regarding whether senile macular degeneration, age-related macular degeneration and 'yellow spot' were the same disease. They expressed a desire to know more about the disease and its consequences. They particularly requested time for receiving information, and the opportunity to discuss it, as in the focus groups. This target group need a health education programme based on their own perceptions. The main issues in such a programme should be to convey information, teach ADL strategies, provide support and foster problem-solving.

Prerectal peritoneal pouch with herniating small bowel -- a unique cause of severe constipation in a child.

We report a 14-year-old boy with severe constipation with a four-year history of no spontaneous bowel movements. Defecography, with synchronously administered barium enema and barium meal, showed that the small bowel could slide down in front of the rectum obstructing defecation. He was operated and a prerectal peritoneal pouch was found, which was resected. Postoperatively he has had daily spontaneous bowel movements. We have found no similar case described in the literature.

Ultrafiltration failure and dialysate glucose in CAPD.

This study was performed to evaluate the use of the glucose concentration versus glucose absorption in the dialysate to estimate ultrafiltration efficacy. In 26 patients (16 men, 10 women) on continuous ambulatory peritoneal dialysis, a total of 128 dwells were investigated. Each dwell lasted 5 hours, using 2 L of peritoneal dialysis fluid containing 140 mmol/L glucose. After the dwell, the removed peritoneal dialysis fluid was weighted. The concentrations of creatinine and glucose and the total amount of glucose were analyzed. The mean concentration of dialysate glucose for the whole group after a 5-hour dwell was 38.9 +/- 8.9 (SD) mmol/L. The ultrafiltration volume was inversely correlated to the glucose absorbed (r = -0.59, p = 0.002), but was not significantly correlated to the glucose concentration in the dialysate (r = 0.40, p = 0.057). The ultrafiltration volume was not related to the creatinine concentration in the dialysate or the body weight. The average dialysate glucose did not change during a one-year observation time. Patients with diabetes mellitus had significantly lower glucose absorption than nondiabetics (p = 0.045), and patients with low ultrafiltration efficacy had significantly greater glucose absorption than those with normal and high ultrafiltration (p = 0.038). There was no difference between those who had those who had not suffered from peritonitis. The results indicate that the glucose absorption from the dialysis fluid is the best parameter to calculate the peritoneal ultrafiltration efficacy and not the glucose concentration currently used.

The course of renal amyloidosis with uraemia.

The course of uraemia in 47 patients with renal amyloidosis has been studied. The median survival with a S-creatinine of 150 mumol/l was approximately 18 months, but 15% survived for more than 3 years. Deterioration arose often suddenly and was of unclear cause. Approximately 25% of the patients improved for a while and the reason for this temporary improvement is also unclear. The unsteady course during the uraemia period for amyloidosis patients makes prognosis calculations very much less accurate than for patients with cystic kidneys.

Torque reaction in angled nutrunners.

In the process of tightening a joint, the angled nutrunner acts on the operator with a torque reaction in the final stages of the tightening sequence. In the following series of experiments, the torque reaction of a machine acting on various joints is studied. A distinctive feature of the torque reaction is that the handle of the angled nutrunner causes a rapid displacement. The amplitude and time aspect of this displacement depend on the stiffness of the joint (whether it is hard or soft), and the movement of the handle depends on how the operator is holding it. How the operator responds to the torque reaction depends upon, among other things, the displacement amplitude as well as the torque level of the machine. Because of this, the displacement amplitude can be used as a measure of the operator's discomfort.

Orthodontic care in the Swedish Public Dental Service, county of Västerbotten.

The extent of orthodontic care provided by the children's dentist in collaboration with the specialist in the Public Dental Service in Sweden has been surveyed in a northern county (55,000 sq km, 230,000 inhabitants and a university town with 80,000 inhibitants). All (925) children born in 1962, resident in a coastal, an inland or a mountain municipality, had received regular dental care. Orthodontic treatment up to and including 1976 has been noted from their dental service records. About 35 per cent of the children in the coastal and inland areas had needed and received some form of orthodontic treatment. In the mountain area only 2/3 of the children who needed treatment had received it; this was due to shortage of specialists. The continuing needs of 102 of the patients have been registered at a follow-up examination. The treatment need of the children was used as a measure of the success rate and the need was on average low. The ability of the general practitioner to determine the right time for treatment appears good, as does the capacity for giving treatment in collaboration with the specialist. Further orthodontic training for the general practitioner is recommended in order to maintain a high level of orthodontic ability. Orthodontic facilities in the mountain area ought to be strengthened.

A three-dimensional photographic method for documentation and measurement of dental conditions.

A 3-D photographic technique was developed for the registration of bite conditions. This technique may replace plaster models for archiving purposes. To achieve this, photographic equipment and computer programs should be user friendly, not too expensive, and allow measurements that satisfy scientific demands. This purpose was fulfilled by combining the following elements: a photographic unit with standardized conditions for 3-D photography and a reference object with known coordinates where bite impressions or models are placed during photography. To reconstruct 3-D coordinates from photographs these are placed on a digitizer connected to a computer programmed for reconstructing 3-D coordinates from the digitized 2-D data. The accuracy of the 3-D reconstruction of coordinates, x, y, z, was tested by means of 3-D photographs of a test object with reference points having a precision of +/- 0.5 micron. The mean error for distances of 5 mm and 15 mm varied between -0.17 mm and +0.15 mm. Measurements of 45 degrees and 90 degrees angles had an accuracy that varied from -0.5 degree to +0.9 degree and from -1.2 degrees to +1.5 degrees respectively. An angle between lines and a plane of 54.7 degrees displayed a mean error of +/- 1.6 degrees. The test of the accuracy of the 3-D photographic technique for calculating distances and angles demonstrated that it fulfills demands for scientific applications on clinical material.