Early events in hepatitis B infection: the role of inoculum dose

The relationship between the inoculum dose and the ability of the pathogen to invade the host is poorly understood. Experimental studies in non-human primates infected with different inoculum doses of hepatitis B virus have shown a non-monotonic relationship between dose magnitude and infection outcome, with high and low doses leading to 100% liver infection and intermediate doses leading to less than 0.1% liver infection, corresponding to CD4 T-cell priming. Since hepatitis B clearance is CD8 T-cell mediated, the question of whether the inoculum dose influences CD8 T-cell dynamics arises. To help answer this question, we developed a mathematical model of virus–host interaction following hepatitis B virus infection. Our model explains the experimental data well, and predicts that the inoculum dose affects both the timing of the CD8 T-cell expansion and the quality of its response, especially the non-cytotoxic function. We find that a low-dose challenge leads to slow CD8 T-cell expansion, weak non-cytotoxic functions, and virus persistence; high- and medium-dose challenges lead to fast CD8 T-cell expansion, strong cytotoxic and non-cytotoxic function, and virus clearance; while a super-low-dose challenge leads to delayed CD8 T-cell expansion, strong cytotoxic and non-cytotoxic function, and virus clearance. These results are useful for designing immune cell-based interventions.     

Unidirectional dominance of cytoplasmic inheritance in two genetic crosses of Plasmodium falciparum.

Malarial parasites have two highly conserved cytoplasmic DNA molecules: a 6-kb tandemly arrayed DNA that has characteristics of a mitochondrial genome, and a 35-kb circular DNA that encodes functions commonly found in chloroplasts. We examined the inheritance pattern of these elements in two genetic crosses of Plasmodium falciparum clones. Parent-specific oligonucleotide probes and single-strand conformation polymorphism analysis identified single nucleotide changes that distinguished the parental 6- and 35-kb DNA molecules in the progeny. In all 16 independent recombinant progeny of a cross between a Central American clone, HB3, and a Southeast Asian clone, Dd2, the 6- and 35-kb DNAs were inherited from the Dd2 parent. In all nine independent recombinant progeny of a cross between clone HB3 and a likely African clone, 3D7, the 6-kb DNA was inherited from the 3D7 parent. Inheritance of cytoplasmic genomes of the Dd2 and 3D7 parents was, therefore, dominant over that of the HB3 parent. Cytoplasmic DNA molecules were found almost exclusively in the female gametes of malarial parasites; hence, clone HB3 did not appear to have served as a maternal parent for the progeny of two crosses. Defective differentiation into male gametes by clone Dd2 is likely to be a reason for the cytoplasmic inheritance pattern seen in the HB3 x Dd2 cross. However, incompetence of male or female gametes is unlikely to explain the uniparental dominance in recombinant progeny of the HB3 x 3D7 cross, since both parents readily self-fertilized and completed the malaria life cycle on their own. Instead, the data suggest unidirectional parental incompatibility in cross-fertilization of these malarial parasites, where a usually cosexual parental clone can participate only as a male or as a female. Such an incompatibility may be speculated as indicating an early phase of reproductive isolation of P. falciparum clones from different geographical regions.     

Lithium Chloride and Trypan Blue Induce Abnormal Morphogenesis by Suppressing Cell Population Growth

Full primitive streak stage chick embryos were cultured in vitro for 20 hrs and monitored every 4 hr for morphology, cell number and blastoderm area. In normal embryos, the cell population growth is exponential and correlates directly with Increasing morphological rank. The chick blastoderm area expands in two waves, one immediately after gastrulation and another after 16 hr in culture, while cell population growth is predominant between 4–16 hr. Trypan blue and LiCI inhibit cell population growth, epiboly and shaping of organ primordia. Both teratogens induce a similar spectrum of abnormalities although the severity of abnormal development is greater with LiCl for the given dose. In most abnormal embryos the cell population size and blastoderm area are inhibited most, which is detectable already after 12 hr of culture. We have established that the cell population growth, morphogenesis and area expansion constitute a parametric hierarchy with the cell population growth as the most independent parameter in regulating normal morphogenesis.     

Purification and properties of two exo-cellobiohydrolases from a cellulolytic culture of Fusarium lini

Two distinct exo-cellobiohydrolases (1,4-β-d-glucan cellobiohydrolase, EC 3.2.1.91) have been isolated from culture filtrates of Fusarium lini by repeated ammonium sulphate fractionation and isoelectric focusing. The purified enzymes were evaluated for physical properties, kinetics and the mechanism of their action. The results of this work were as follows. (1) A two-step enzyme purification procedure was developed, involving isoelectric focusing and ammonium sulphate fractionation. (2) Yields of pure cellobiohydrolases I and II were 45 and 36 mg l^?1 of culture broth, respectively. (3) Both enzymes were found to be homogeneous, as determined by ultracentrifugation, isoelectric focusing, electrophoresis in polyacrylamide gels containing SDS and chromatography on Sephadex. (4) The molecular weights of the two cellobiohydrolases, as determined by gel filtration and SDS gel electrophoresis, were 50 000–57 000. (5) Both cellobiohydrolases had low viscosity-reducing and reducing sugar activity from carboxymethyl cellulose and high activity with Walseth cellulose and Avicel. (6) The enzymes produced only cellobiose as the end product from filter paper and Avicel, indicating that they are true cellobiohydrolases. (7) Cellobiohydrolase I hydrolysed d-xylan whereas cellobiohydrolase II was inactive towards d-xylan. (8) There was a striking synergism in filter paper activity when cellobiohydrolase was supplemented with endo-1,4-β-d-glucanase [cellulase, 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and β-d-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21).     

Responses of the neurosecretory cells of the freshwater snail,Indoplanorbis exustus to hydrothermal stress

Changes in the neurosecretory cell cytology of I. exustus subjected to hypertonic saline (0.1 ml of 1.5%/snail) loading and thermal stress (35°C) for two hours, have been investigated. Of the two types of neurosecretory cells A and B that are present in the central nervous system (CNS) of I. exustus, striking changes were evident only in B cells. After both treatments, there was about 33% decline in NSM (Neurosecretory material) intensity. However, the nuclear diameter of B cells was significantly (P < 0.001) increased in the snails administrated with hypertonic saline unlike in those exposed to 35°C wherein significant (P < 0.005) decline was evident. The adaptive significance of the neuroendocrine system of I exustus is discussed in relation to hydrothermal stress.     

Mycoplasmal infection of lymphocyte cell cultures: Infection withM. salivarium

Summary Many conclusions concerning cell culture mycoplasmas are based on data from studies in fibroblast cultures. Some conclusions may not be valid in other types of differentiated cell cultures.M. salivarium was isolated from 35 human lymphocyte cultures (HLC), 34 from the same laboratory. The organism grew to more than 10⁸ colony forming units (CFU) per ml of lymphocyte suspensions and was readily detectable by microbiological culture, uridine phosphorylase, and uridine/uracil assays. Direct mycoplasmal assays on HLC by DNA fluorochrome staining and scanning electron microscopy (SEM) yielded artifacts that interfered with diagnosis. For DNA and SEM of HLC, inoculation into indicator cell cultures is recommended.M. salivarium infection of HLC did not produce any immediate difference in growth rates; however, infected cultures eventually died 14 to 29 passages after infection in contrast to uninfected controls. The same organism in 3T6 fibroblasts effected a 60% decrease in growth rate. AlthoughM. salivarium is a frequent isolate from the oral cavity, it is a rare cell culture isolate.M. salivarium was able to initiate growth over a wide pH range, grew as well in cell cultures as in cell-free media, and was resistant to 50 μg per ml of gentamycin, tylocine, kanamycin, and erythromycin. By C₀t_1/2 analysis,M. salivarium had a genomic molecular weight of 4.2×10⁸ daltons.M. salivarium did not increase chromosome aberrations in one HLC. Some of these results have application to infection of HLC by other mycoplasmal species. These studies were supported by contracts NO1-AG-82117 from the National Institute on Aging, NO1-GM-9-2101 from the National Institute of General Medical Sciences, and Grant RO1-A1-15748 from the National Institute of Allergy and Infectious Diseases.