Quantitation of hepatitis B viral DNA by solution hybridization: Comparison with DNA polymerase and hepatitis B e antigen during antiviral therapy

Serological markers of hepatitis B virus (HBV) replication were assessed in a randomized, controlled trial of prednisone withdrawal followed by α -interferon in the treatment of chronic hepatitis B. HBV DNA levels in more than 700 serial serum samples from 41 patients were determined by a sensitive and quantitative solution hybridization assay. Results were compared with HBV DNA polymerase (DNAp) activity and hepatitis B e antigen (HBeAg) in 21 untreated controls and 20 treated patients. Among treated patients, the mean pretherapy HBV DNA values were higher in nonresponders than in responders. During prednisone treatment, DNA levels increased an average of 2.1-fold in responders and 1.4-fold in nonresponders. During the 2-week rest interval between prednisone and interferon, DNA values fell an average of 57% in responders. In contrast, the mean DNA values in nonresponders did not change during the same interval. This early distinction between responders and nonresponders was not apparent from DNAp or HBeAg results. During interferon treatment, HBV DNA became undetectable in responders and remained negative during a 1-year follow-up. DNA in nonresponders declined to 14% of baseline during interferon treatment but increased to pretherapy levels after treatment. DNAp values generally paralleled HBV DNA values, but DNAp activity showed more variability and lower sensitivity than did the hybridization assay results. HBeAg values varied independently of HBV DNA and DNAp with a much delayed decline in responders. These results indicate that HBV DNA, when measured quantitatively by a sensitive solution hybridization assay, is an early predictor of the effects of antiviral agents on replication.     

Monocyte chemotactic protein-3 (MCP3) interacts with multiple leukocyte receptors: binding and signaling of MCP3 through shared as well as unique receptors on monocytes and neutrophils

The diversity of monocyte chemotactic protein (MCP)3 target cell types, as well as the capacity of MCP3 to desensitize leukocyte responses to other CC chemokines, suggested that MCP3 may interact with multiple CC chemokine receptors. The purpose of this study is to establish how MCP3 binds and activates monocytes and neutrophils. We show that human monocytes exhibit high-affinity binding for ¹²⁵I-MCP3 with an estimated Kd of 1–3 nM and about 10000 binding sites/cell. The binding of ¹²⁵I-MCP3 to monocytes was progressively less well competed by CC chemokines macrophage inflammatory protein (MIP)lα (Kd = 5–10 nM), RANTES (Kd = 5–10 nM), MCP1 (monocyte chemoattractant and activating factor, or MCAF) (Kd = 60 nM) and MIP1β (Kd > 100 nM). On the other hand, unlabeled MCP3 displaced the binding of radiolabeled MIP1α, RANTES, MCP1 and MIP1β as effectively as the isologous CC chemokines. In agreement with the binding data, pretreatment of monocytes with MCP3 completely desensitized the calcium flux in response to MIP1α and RANTES. However, MIP1α and RANTES failed to desensitize the response of monocytes to MCP3. MCP3 and MCP1 partially desensitized each other's effects on monocytes. These binding and cross-desensitization results suggest that MCP3 binds and signals through other binding sites in addition to those shared with MIP1α, RANTES and MCP1. The unidirectional competition for MIP1β binding and signaling by MCP3 suggests the existence of an as-yet unidentified site for MCP3 shared with MIP1β. The existence of another unique binding site(s) for MCP3 was further shown by the failure of any of the other CC chemokines to compete effectively for MCP3 binding on neutrophils. MCP3 in our study was also the only human CC chemokine that consistently chemoattracted neutrophils. These results suggest that MCP3 is a ligand that can bind and activate a broad range of target cells through receptors shared by other CC chemokines as well as its own receptor.     

Endothelial cells inhibit receptor-mediated superoxide anion production by human polymorphonuclear leukocytes via a soluble inhibitor

Confluent monolayers of bovine pulmonary artery endothelial cells (BPAE) or human umbilical vein endothelial cells (HUVE) inhibited by 80 to 90% the production of O2- by added human neutrophils (PMNs) stimulated by plasma membrane receptor-mediated activators (formylmethionylleucylphenylalanine [fMLP], opsonized zymosan, heat-killed Staphylococci), but not by non-plasma membrane receptor-mediated activators (phorbol myristate acetate and delta-hexachlorocyclohexane). Degranulation induced by fMLP was also inhibited by BPAE. Inhibition was not affected by eicosatetraynoic acid (ETYA) or indomethacin. To assess the role of cell-cell contact, 0.45-microns-pore culture plate inserts were employed to prevent PMN-endothelial cell contact during incubation. A similar amount of inhibition of stimulated PMNs superoxide production was seen as compared to PMN-endothelial incubations where contact occurred. A soluble component released by BPAE monolayers, when added to PMNs, duplicated the inhibition seen by BPAE-PMN co-incubation. Incubation of BPAE with adenosine deaminase did not reduce inhibition of O2- production compared to controls without adenosine deaminase. There was no evidence of endothelial scavenging of O2- generated by hypoxanthine-xanthine oxidase, and inhibition of endothelial superoxide dismutase did not diminish the inhibitory effort. We conclude that cell contact is not required for BPAE inhibition of fMLP-stimulated O2- production by PMN, and that scavenging of superoxide anion is not the mechanism. The inhibitor appears to be a polypeptide with an apparent molecular weight between 1,000 and 10,000 D and does not appear to be adenosine, an arachidonate metabolite, or superoxide dismutase. The mechanism may involve down-regulation of plasma membrane receptor-mediated activation of PMNs.     

Identification of two mutations in a compound heterozygous child with dihydrolipoamide dehydrogenase deficiency

An infant girl with elevated blood lactate, pyruvate, and plasma branched-chain amino acids was diagnosed with dihydrolipoamide dehydrogenase (E3; dihydrolipoamide: NAD+ oxidoreductase, EC 1.8.1.4) deficiency. Activities of the pyruvate dehydrogenase complex and E3 from patient were 26 and 2% of controls in blood lymphocytes, and 11 and 14% in cultured skin fibroblasts, respectively. Western blot analysis demonstrated that the amount of E3 protein in fibroblasts from the patient and her father was about half of controls, while Northern blot analysis showed normal amounts of E3 RNA. DNA sequencing of cloned full-length E3 cDNAs from the patient revealed two mutations in separate alleles. One is a single base insertion of an extra adenine in the last codon of the leader peptide sequence (TAC-->TAAC) leading to a nonsense mutation which results in the premature termination of the precursor E3 polypeptide (Y35X). The other is a missense mutation due to substitution of guanine for adenine, causing an Arg-->Gly substitution at amino acid 460 of the mature protein (R460G) which triggers the loss of E3 activity probably by structural change in the E3 dimer. DNA sequencing of E3 cDNAs from the parents demonstrated that the nonsense mutation was inherited from the father and the missense mutation was inherited from the mother.      

Tula Virus as Causative Agent of Hantavirus Disease in Immunocompetent Person,Germany

We report molecular evidence of Tula virus infection in an immunocompetent patient from Germany who had typical signs of hantavirus disease. Accumulating evidence indicates that Tula virus infection, although often considered nonpathogenic, represents a threat to human health.     

Facet coverage in children on flexion lateral cervical radiographs

STUDY DESIGN: This radiographic study was designed to attempt to develop standards of facet coverage (overlap) on lateral cervical radiographs during voluntary flexion. OBJECTIVE: To produce normative standards for minimum facet coverage in children. SUMMARY OF BACKGROUND DATA: Previous studies on normative standards of facet coverage have been performed only in adults. METHODS: Thirty-six children with minor trauma had lateral flexion-extension radiographs. A standard filming sequence was used in all. Facet joint overlap at each level was divided by the anteroposterior diameter of the upper adjacent cervical body at each level. RESULTS: On linear regression analysis, these ratios did not vary significantly with age at C2-C3 through C6-C7. Means and standard deviations were determined for C2-C7. CONCLUSIONS: These ratios may prove useful in evaluation of children with possible ligamentous injury.     

A simple method for production of slides of CT images from multiformat radiographs.

Sixteen on 1 multiformat images of CT scans can be mounted directly into special "super slide" 2 by 2 in. mounts. Use of special photographic equipment is thus avoided.     

Role of radiology in the treatment of malignant hilar biliary strictures 2: 10 years of single‐institution experience with percutaneous treatment

We reviewed the results of percutaneous intervention of hilar biliary malignancy over a 10‐year period at a single institution: the Royal Melbourne Hospital. Ninety‐nine patients (100 treated in total) were included. Information was retrieved by retrospective examination of patient notes and radiology, combined with interviews with family and relevant physicians. Sixty‐nine patients were treated with insertion of semipermanent stents, 19 had external drain tubes, and 25 received percutaneous access for Iridium brachytherapy. Adequate drainage was achieved in 87% of the patients stented, and percutaneous access was successful in 96% of patients planned for brachytherapy. Of those patients undergoing endoprosthesis insertion, early complications occurred in 39% and late complications in 23%. Average survival for the entire patient population was 227.3 days, with a median of 167 days. Longer survival times (213 vs 142 days) and lower complication rates (44 vs 64%) are observed with metal stents in comparison with plastic stents. Percutaneous intervention is an important treatment option in hilar biliary malignancy, particularly in patients unfit for surgery. Reasonable survival with good palliation is the most common outcome, and most patients do not require further intervention.