Hepatitis B virus genetic diversity and its impact on diagnostic assays

Summary.  Hepatitis B virus (HBV) circulates in blood as closely related, but genetically diverse molecules called quasispecies. During replication, HBV production may approach 10¹¹ molecules/day, although during peak activity this rate may increase 100–1000 times. Generally, DNA polymerases have excellent fidelity in reading DNA templates because they are associated with an exonuclease which removes incorrectly added nucleotides. However, the HBV-DNA polymerase lacks fidelity and proofreading function partly because exonuclease activity is either absent or deficient. Thus, the HBV genome and especially the envelope gene, is mutated with unusually high frequency. These mutations can affect more than one open reading frame because of overlapping genes. The S gene contains an exposed major hydrophilic region (residues 110–155), which encompasses the 'a' determinant that is important for inducing immunity. Nucleotide substitutions in this region are common and result in reduced binding or failure to detect hepatitis B surface antigen (HBsAg) in diagnostic assays. Adaptive immunity also depends on the recognition of HBsAg by specific antibody and variants pose a threat if they interfere with binding to antibody. Finally, genomic hypervariability allows HBV to escape selection pressures imposed by antiviral therapies, vaccines and the host immune system, and is responsible for creating genotypes, subgenotypes and subtypes.     

Response to a hepatitis B polypeptide vaccine in micelle form in a young adult population

Polypeptide micelles with relative molecular weights of 25,000 (p25) and 30,000 (gp30) daltons were prepared from native 22-nm hepatitis B surface antigen (HBsAg) particles. This p25/gp30 complex was alum-adsorbed, and three dosage levels (20 micrograms, 4 micrograms, and 0.8 micrograms) were administered at 0, 1, and 6 months to 51 human volunteers. Local and systemic reactions were clinically insignificant, and all vaccinees seroconverted, regardless of dose. As anticipated, antibody responses diminished as the dosage was reduced. Seroconversion rates and geometric mean antibody levels for the 20 micrograms dosage group were significantly better than those observed with a commercial vaccine and were comparable to those achieved after immunization with 40 micrograms of the intact 22-nm particles used to prepare the polypeptides. By 2 weeks, an anti-HBs response was elicited in 80% of the group receiving 20 micrograms of the polypeptide vaccine. This rapid response to immunization may be particularly beneficial for postexposure prophylaxis where the early development of immunity is advantageous.     

Immunological and biophysical properties of hepatitis B antigen labeled by the chloramine-T and by the lactoperoxidase methods.

Optimal conditions were sought for the radiolabeling of microgram quantities of hepatitis B surface antigen (HBs Ag) employing the chloramine-T or lactoperoxidase iodination procedures. Preparations of HBsAg labeled by these procedures are referred to as chloramine-T preparations and lactoperoxidase preparations, respectively. Labeled HBsAg having specific activities between 10-20 muCi/mug were found to display the greatest degree of sensitivity for unlabeled HBsAg and for anti-HBs using a double-antibody radioimmunoassay (RIA-DA). Increasing the specific activity above this level redulted in a decreased affinity of labeled 1251-HBs Ag for anti-HBs, indicating that soluble antigenic alterations had developed. At equivalent specific activities, chloramine-T preparations competed less effectively for unlabeled HBs Ag than lactoperoxidase preparations, and anti-HBs endpoint titers were slightly reduced, especially among preparations of high specific activity (greater than or equal to 65 muCi/mug). Chloramine-T preparations of HBs Ag (sp. act. 15--30 muCi/mug) showed essentially no antigenic deterioration over a 2-month period at minus 196 degrees C or minus 70 degrees C. Utilization of optimally labeled 1251-HBs Ag has increased the sensitivity of the RIA-DA for unlabeled HBs Ag 30-fold to a level below 1 ng/ml and enhanced antiamine-T method revealed that only the most acidic population was labeled (pH 3.75+/-0.5). In contrast, six antigenic components with distinct pI values ranging from 3.7 to 5.2 were detected by RIA-DA in both unlabeled HBs ag and in the chloramine-T preparation. This indicated that the chloramine-T method did not radically change the relative number or charge of each of the pI populations present in purified preparations of HBs Ag. Analysis of HBs Ag iodinated by the lactoperoxidase procedure revealed the presence of three of four populations of particles with pI values ranging from 3.9 to 4.5, suggesting that this procedure labels HBs Ag more uniformly.     

Immune-complex glomerulonephritis, intranuclear particles in hepatocytes, and in vivo clearance rates in sub-human primates inoculated with HBs AG-containing plasma.

Twelve subhuman primates [eight baboons (Papio anubis) and four rhesus (Macaca mulatta)] were inoculated with varying amounts of human plasma containing HB_s Ag with Dane particles. Serial blood specimens and liver, kidney, bone marrow, and lymph node biopsies were obtained from each animal over 10–12 mo. No measurable evidence of HB_s Ag production was observed in any animal after the initial clearance of antigen, although the presence of anti-HB_s in most of the animals studied might have precluded its detection. Two baboons developed progressive focal glomerulonephritis with mesangial alterations over a 4- to 10-mo period of time. Liver biopsies from these baboons and one rhesus monkey revealed the presence of 26-nm intranuclear particles in the hepatocytes at 4 mo postinoculation. The intranuclear particles were similar in morphology and in size to the cores of Dane particles. Kinetic analysis of antigen clearance implied a rapid uptake of HB_s Ag from the blood to the liver and spleen over an 8 to 24-hr period of time and an in vivo clearance rate ($\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) of 28.2 hr.     

The physiological response of protease inhibition in dystrophic muscle

Duchenne muscular dystrophy (DMD) is caused by the production of a non‐functional dystrophin gene product and a failure to accumulate functional dystrophin protein in muscle cells. This leads to membrane instability, loss of Ca²⁺ homoeostasis and widespread cellular injury. Associated with these changes are increased protease activities in a variety of proteolytic systems. As such, there have been numerous investigations directed towards determining the therapeutic potential of protease inhibition. In this review, evidence from genetic and/or pharmacological inhibition of proteases as a treatment strategy for DMD is systematically evaluated. Specifically, we review the potential roles of calpain, proteasome, caspase, matrix metalloproteinase and serine protease inhibition as therapeutic approaches for DMD. We conclude that despite early results to the contrary, inhibition of calpain proteases is unlikely to be successful. Conversely, evidence suggests that inhibition of proteasome, matrix metalloproteinases and serine proteases does appear to decrease disease severity. An important caveat to these conclusions, however, is that the fundamental cause of DMD, dystrophin deficiency, is not corrected by this strategy. Hence, this should not be viewed as a cure, but rather, protease inhibitors should be considered for inclusion in a therapeutic cocktail. Physiological Relevance. Selective modulation of protease activity has the potential to profoundly change intracellular physiology resulting in a possible treatment for DMD. However, alteration of protease activities could also lead to worsening of disease progression by promoting the accumulation of substrates in the cell. The balance of benefit and potential damage caused by protease inhibition in human DMD patients is largely unexplored.     

Diagnostik und Therapie der ligamentären Ellenbogeninstabilitäten

Posttraumatic or acute instability of the elbow can develop after complete or incomplete dislocation of the elbow. Isolated medial collateral ligament ruptures can be a result of valgus trauma and lateral collateral ligament ruptures of pivot shift or varus trauma. Chronic instability of the elbow may occur after conservative or operative treatment of a traumatic ligament rupture without stable healing. Repetitive overuse in sports or professions may also cause chronic instability. This article provides the reader an overview about current concepts in elbow instability.