Mathematical model comparisons of potential non-typeable Haemophilus influenzae vaccine effects

Vaccines to prevent acute otitis media (AOM) caused by non-typeable Haemophilus influenzae (NTHi) are under development. Because NTHi is highly variable and colonization rates are high, special vaccine characteristics and trial designs might be needed. We examined in mathematical models the equilibrium NTHi-caused AOM rate given hypothetical vaccines that generated immunity identical to corresponding maximal naturally acquired immunity. Vaccines were examined with single effects and combinations of immunity affecting (1) AOM rates given colonization (pathogenicity), (2) susceptibility to colonization, and (3) contagiousness given colonization. Percent reductions in AOM across all preschool children were (1) 34%, (2) 31%, (3) 9%, (1 and 2) 57%, (2 and 3) 50%, and (1, 2, and 3) 75%. Effects on children in daycare vs. not in daycare were (1) 18 vs. 48%, (2) -1 vs. 57%, (3) 13 vs. 5%, (1 and 2) 30 vs. 79%, (2 and 3) 33 vs. 60%, and (1, 2, and 3) 64 vs. 85%. Pure pathogenicity effects (1 alone) will need to be supplemented by transmission effects. The effects of susceptibility (2 alone) are diminished or negative because children protected against colonization have lower levels of immunity to (1) and (3) than unvaccinated children. For trials to predict population effects, both colonization and AOM outcomes must be studied and all three effects must be evaluated. This need arises because, unlike H. influenzae type B, high NTHi exposure diminishes cumulative vaccine effects and high colonization rates generate rapid accumulation of natural immunity that alters the indirect effects of vaccine immunity on transmission differently by age and daycare status.     

Upregulation of Ca2+ removal in human skeletal muscle: a possible role for Ca2+-dependent priming of mitochondrial ATP synthesis

In muscle, ATP is required for the powerstroke of the myosin head, the detachment of actin and myosin filaments, and the reuptake of Ca²⁺ into the sarcoplasmic reticulum. During contraction-relaxation, large amounts of ATP are consumed at the sites of action of the myosin-ATPase and sarcoplasmic reticulum Ca²⁺-ATPase. The present study addresses the consequences of a reduction in mitochondrial ATP production capacity on sarcoplasmic Ca²⁺ handling. To this end, myotubes were cultured from patient quadriceps with a biochemically defined decrease in the maximal rate of mitochondrial ATP production and were loaded with indo 1 for imaging of sarcoplasmic Ca²⁺ changes in real time by confocal microscopy. Myotubes were field-stimulated with 10-ms pulses of 16 V to evoke transient rises in sarcoplasmic Ca²⁺ concentration ([Ca²⁺]_S). Three single pulses, two pulse trains (1 Hz), and one single pulse were applied in succession to mimic changing workloads. Control myotubes displayed [Ca²⁺]_S transients with an amplitude that was independent of the strength of the stimulus. Intriguingly, the rate of sarcoplasmic Ca²⁺ removal (CRR) was significantly upregulated during the second and subsequent transients. In myotubes with a reduced mitochondrial ATP production capacity, the amplitude of the [Ca²⁺]_S transients was markedly increased at higher stimulus intensities. Moreover, upregulation of the CRR was significantly decreased compared with control. Taken together, these results are in good agreement with a tight coupling between mitochondrial ATP production and sarcoplasmic Ca²⁺ handling. Moreover, they support the existence of a relatively long-lasting mitochondrial memory for sarcoplasmic [Ca²⁺] rises. This memory, which manifested itself as an increase in CRR upon recurrent stimulation, was impaired in patient myotubes with a reduced mitochondrial ATP production capacity. sarcoplasmic Ca²⁺ removal; video-rate imaging; indo 1; electrical stimulation; mitochondrial memory     

AFLP markers as a tool to reconstruct complex relationships: A case study in Rosa (Rosaceae)

The genus Rosa has a complex evolutionary history caused by several factors, often in conjunction: extensive hybridization, recent radiation, incomplete lineage sorting, and multiple events of polyploidy. We examined the applicability of AFLP markers for reconstructing (species) relationships in Rosa, using UPGMA clustering, Wagner parsimony, and Bayesian inference. All trees were well resolved, but many of the deeper branches were weakly supported. The cluster analysis showed that the rose cultivars can be separated into a European and an Oriental cluster, each being related to different wild species. The phylogenetic analyses showed that (1) two of the four subgenera (Hulthemia and Platyrhodon) do not deserve subgeneric status; (2) section Carolinae should be merged with sect. Cinnamomeae; (3) subsection Rubigineae is a monophyletic group within sect. Caninae, making sect. Caninae paraphyletic; and (4) there is little support for the distinction of the five other subsections within sect. Caninae. Comparison of the trees with morphological classifications and with previous molecular studies showed that all methods yielded reliable trees. Bayesian inference proved to be a useful alternative to parsimony analysis of AFLP data. Because of their genome-wide sampling, AFLPs are the markers of choice to reconstruct (species) relationships in evolutionary complex groups.     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

Use of Combusted Natural Gas to Cultivate the Anaerobic Bacterial Flora from the Cecum Contents of Mice

The use of combusted natural gas as atmosphere for cultivation of the anaerobic bacterial flora from the cecum of mice is described. The combusted gas (inert gas) continuously flushes a glove box in which it is possible to cultivate a number of strict anaerobes from the cecum of mice.     

Membrane-initiated Ca(2+) signals are reshaped during propagation to subcellular regions.

An important aspect of Ca(2+) signaling is the ability of cells to generate intracellular Ca(2+) waves. In this study we have analyzed the cellular and subcellular kinetics of Ca(2+) waves in a neuroendocrine transducer cell, the melanotrope of Xenopus laevis, using the ratiometric Ca(2+) probe indo-1 and video-rate UV confocal laser-scanning microscopy. The purpose of the present study was to investigate how local Ca(2+) changes contribute to a global Ca(2+) signal; subsequently we quantified how a Ca(2+) wave is kinetically reshaped as it is propagated through the cell. The combined kinetics of all subcellular Ca(2+) signals determined the shape of the total cellular Ca(2+) signal, but each subcellular contribution to the cellular signal was not constant in time. Near the plasma membrane, [Ca(2+)](i) increased and decreased rapidly, processes that can be described by a linear and exponential function, respectively. In more central parts of the cell slower kinetics were observed that were best described by a Hill equation. This reshaping of the Ca(2+) wave was modeled with an equation derived from a low-pass RC filter. We propose that the differences in spatial kinetics of the Ca(2+) signal serves as a mechanism by which the same cellular Ca(2+) signal carries different regulatory information to different subcellular regions of the cell, thus evoking differential cellular responses.     

Efforts to broaden HIV-1-specific immunity by boosting with heterologous peptides or envelope protein and the influence of prior exposure to virus

In two previous studies, we have demonstrated the successful protection of human immunodeficiency virus type 1 (HIV-1)-vaccinated rhesus macaques from challenge with SHIV(SF13) with envelop immunogens derived from the closely related HIV-1(SF2) strain. Here we report on two follow-up studies in which we aimed to broaden immunity in order to elicit protection from a more diverse heterologous challenge with SHIV(SF33). In the first study, animals were boosted once with HIV-1(SF33) V2 and V3 peptides that were cross-linked to influenza immune-stimulating complexes (ISCOMs). In the second study, monkeys were boosted twice at 12-week intervals, using a heterologous recombinant gp120 derived from HIV-1(SF33) that was either incorporated into ISCOMs or mixed with the MF59 adjuvant. In both studies, the animals were challenged with 50 monkey infectious doses of SHIV(SF33) 4 weeks after the final boost. All controls became readily infected with the heterologous challenge virus SHIV(SF33). Neither boosting with heterologous SF33 peptides or gp120 afforded protection from infection to SF2-vaccinated animals that had previously resisted SHIV(SF13) challenge. These results demonstrate the importance of developing vaccine strategies that are capable of generating broad immune responses early in the immunization protocol. Furthermore, these findings may illustrate the potential pitfalls of early antigenic sin.