A Novel Assay for Antibody-Dependent Cell-Mediated Cytotoxicity against HIV-1- or SIV-Infected Cells Reveals Incomplete Overlap with Antibodies Measured by Neutralization and Binding Assays

The resistance of human immunodeficiency virus type 1 (HIV-1) to antibody-mediated immunity often prevents the detection of antibodies that neutralize primary isolates of HIV-1. However, conventional assays for antibody functions other than neutralization are suboptimal. Current methods for measuring the killing of virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC) are limited by the number of natural killer (NK) cells obtainable from individual donors, donor-to-donor variation, and the use of nonphysiological targets. We therefore developed an ADCC assay based on NK cell lines that express human or macaque CD16 and a CD4⁺ T-cell line that expresses luciferase from a Tat-inducible promoter upon HIV-1 or simian immunodeficiency virus (SIV) infection. NK cells and virus-infected targets are mixed in the presence of serial plasma dilutions, and ADCC is measured as the dose-dependent loss of luciferase activity. Using this approach, ADCC titers were measured in plasma samples from HIV-infected human donors and SIV-infected macaques. For the same plasma samples paired with the same test viruses, this assay was approximately 2 orders of magnitude more sensitive than optimized assays for neutralizing antibodies—frequently allowing the measurement of ADCC in the absence of detectable neutralization. Although ADCC correlated with other measures of Env-specific antibodies, neutralizing and gp120 binding titers did not consistently predict ADCC activity. Hence, this assay affords a sensitive method for measuring antibodies capable of directing ADCC against HIV- or SIV-infected cells expressing native conformations of the viral envelope glycoprotein and reveals incomplete overlap of the antibodies that direct ADCC and those measured in neutralization and binding assays.     

Relation of left ventricular mass to QTc in normotensive severely obese patients

Prolongation of the corrected QT interval (QTc) has been described in obese subjects. This study assesses the relation of left ventricular (LV) mass to QTc in normotensive severely obese subjects. Fifty normotensive patients whose BMI was ≥40 kg/m(2) (mean age: 38 ± 7 years) were studied. QTc was derived using Bazett's formula. LV mass was calculated using the formula of Devereux et al. and was indexed to height(2.7). Mean QTc was 428.8 ± 19.0 ms and was significantly longer in those with than in those without LV hypertrophy (P < 0.01) QTc correlated positively and significantly with BMI (r = 0.392, P < 0.025), LV mass/height(2.7) (r = 0.793, P < 0.0005), systolic blood pressure (r = 0.742, P < 0.001), LV end - systolic wall stress (r = 0.746, P < 0.001) and LV internal dimension in diastole (r = 0.788, P < 0.0005). Among five variables tested, LV mass/height(2.7) was identified as the sole predictor of QTc by multivariate analysis. In conclusion, LV mass and loading conditions that may affect LV mass are important determinants of QTc in normotensive severely obese subjects.     

Characterization of a Theta-Type Plasmid from Lactobacillus sakei: a Potential Basis for Low-Copy-Number Vectors in Lactobacilli

The complete nucleotide sequence of the 13-kb plasmid pRV500, isolated from Lactobacillus sakei RV332, was determined. Sequence analysis enabled the identification of genes coding for a putative type I restriction-modification system, two genes coding for putative recombinases of the integrase family, and a region likely involved in replication. The structural features of this region, comprising a putative ori segment containing 11- and 22-bp repeats and a repA gene coding for a putative initiator protein, indicated that pRV500 belongs to the pUCL287 subfamily of theta-type replicons. A 3.7-kb fragment encompassing this region was fused to an Escherichia coli replicon to produce the shuttle vector pRV566 and was observed to be functional in L. sakei for plasmid replication. The L. sakei replicon alone could not support replication in E. coli. Plasmid pRV500 and its derivative pRV566 were determined to be at very low copy numbers in L. sakei. pRV566 was maintained at a reasonable rate over 20 generations in several lactobacilli, such as Lactobacillus curvatus, Lactobacillus casei, and Lactobacillus plantarum, in addition to L. sakei, making it an interesting basis for developing vectors. Sequence relationships with other plasmids are described and discussed.     

Administration of IL-7 to normal mice stimulates B-lymphopoiesis and peripheral lymphadenopathy.

Normal mice were injected with IL-7 (500 ng, twice daily) for various periods of time up to 6 days and the cellularity and phenotypic composition of the thymus, spleen, lymph node, and bone marrow was assessed. After 6 days of treatment, significant increases in the cellularity of the spleen, lymph node, and bone marrow were observed which returned to the normal range within 6 days after cessation of treatment. After 3 days of IL-7 treatment, increased numbers of B220+/surface(s) IgM- bone marrow cells were observed. After 6 days of treatment, these numbers were still further increased and a significant population of B220+/sIgM- cells were observed in the spleen. The numbers of c mu+/sIgM- cells were also increased in the IL-7-treated mice. Analysis of the expression of B220 and BP-1 on the sIgM- bone marrow cells revealed that the B220+/BP-1+ population was dramatically increased after IL-7 treatment and the size of the B220+/BP-1- population did not differ from control mice. The pre-B cell numbers declined rapidly after the cessation of IL-7 treatment. After 6 days of IL-7 treatment, a twofold increase in the number of B cells in the spleen and lymph node was observed. The B cell numbers declined to normal values within 6 days after the cessation of IL-7 administration. In the spleens of the IL-7-treated mice, there was a significant increase in the number of B cells with an immature phenotype (e.g., sIgMhi/sIgDlo, decreased levels of Ia and FcR expression). The numbers of CD8+ and CD4+ T cells were also increased in the lymph node and spleen of the IL-7-treated mice. These numbers declined to normal levels after the cessation of IL-7 treatment.     

The ATPase activity of subfragment-1 from the hypertrophied heart.

Myosin and subfragment-1 were prepared from rabbit hearts hypertrophied secondary to pulmonary artery constriction. The Ca2+ -stimulated ATPase activity was reduced while the potassium/EDTA-stimulated ATPase activity was unchanged in both the myosin and subfragment 1 (S-1) from hypertrophied hearts. When hypertrophy myosin was mixed with an equal quantity of control myosin, the ATPase activity of the mixed protein fell halfway between control and hypertrophy values. Similar results were obtained with control and hypertrophy S-1. The actin-stimulated ATPase activity of hypertrophy S-1 was slightly depressed but unlike hypertrophy myosin this depression was not significant when compared to normal S-1. This suggests that papain cleavage may have removed part of the conformational difference that exists between control and hypertrophy myosins.     

Lactobacillus casei Is Able To Survive and Initiate Protein Synthesis during Its Transit in the Digestive Tract of Human Flora-Associated Mice

Live Lactobacillus casei is present in fermented dairy products and has beneficial properties for human health. In the human digestive tract, the resident flora generally prevents the establishment of ingested lactic acid bacteria, the presence of which is therefore transient. The aim of this work was to determine if L. casei DN-114 001 survives during transit and how this bacterium behaves in the digestive environment. We used the human flora-associated (HFA) mouse model. L. casei DN-114 001 was genetically modified by the introduction of erm and lux genes, encoding erythromycin resistance and luciferase, respectively. For this modified strain (DN-240 041), light emission related to luciferase expression could easily be detected in the contents of the digestive tract. When inoculated into the digestive tract of HFA mice, L. casei (DN-240 041) survives but is eliminated with the same kinetics as an inert transit marker, indicating that it does not establish itself. In pure culture of L. casei, luciferase activities were high in the exponential and early stationary growth phases but decreased to become undetectable 1 day after inoculation. Viability was only slightly reduced even after more than 5 days. After transit in HFA mice, luciferase activity was detected even when 5-day-old L. casei cultures were given to the mice. In culture, the luciferase activity could be restored after 0.5 to 7 h of incubation in fresh medium or milk containing glucose, unless protein synthesis was inhibited by the addition of chloramphenicol or rifampin. These results suggest that in HFA mice L. casei DN-240 041, and thus probably L. casei DN-114 001, is able to initiate new protein synthesis during its transit with the diet. The beneficial properties of L. casei-fermented milk for human health might be related to this protein synthesis in the digestive tract.     

Effects of Nitrogen and Salinity on Growth and Competition Between a Native Grass and an Invasive Congener

Numerous studies show that an increase in the availability of limiting resources can increase invasion by non-native plants into natural communities. One possible explanation is that the ability of natives to compete with non-natives tends to decrease when resource availability is increased. We tested this hypothesis in a competition experiment using two closely matched plant species and two environmental factors related to limiting resources in a coastal grassland system on Bodega Head in northern California. We grew the native grass Bromus carinatus and the non-native grass B. diandrus together and apart at different levels of soil nitrogen crossed with different levels of soil salinity. Both species are abundant in the grassland and previous work suggested that the abundance of B. carinatus is lower and the abundance of B. diandrus is higher on soil that has been enriched with nitrogen. Salinity has been shown to be negatively associated with invasion by B. diandrus into another California grassland, and to vary significantly over short distances in the grassland at Bodega Head, where it could affect water availability, which strongly limits plant growth during the dry season. Contrary to our prediction that low resource availabilities would increase the relative competitive ability of the native, the ability of B. carinatus to compete with B. diandrus was not greater when nitrogen availability was lower or when soil salinity was higher. Instead, high salinity increased the relative competitive ability of the non-native, and low nitrogen had little effect on competition. This suggests that preventing resource enrichment will not suffice to control invasion by non-native plant species in this grassland.     

Physiological integration in an introduced, invasive plant increases its spread into experimental communities and modifies their structure

What determines the invasiveness of introduced plants is still poorly known. Many of the most invasive plant species are clonal, and physiological integration between connected individuals (ramets) of clonal plants may contribute to their ability to spread into communities and reduce performance of existing species. This contribution of integration to the invasiveness of clonal plants may be greater in denser communities. A greenhouse study was conducted to test these two hypotheses. High- and low-density communities were created by sowing seeds of eight grassland species. Each community was planted with three ramets of the stoloniferous, introduced plant Alternanthera philoxeroides that were disconnected from or left connected to ramets growing on bare soil. Connection increased the spread of Alternanthera within a community, but did not reduce community biomass. Alternanthera grew less in high-density communities, but connection did not improve its growth more than in low-density communities. Low-density communities had higher evenness when Alternanthera was connected than when it was disconnected because shoot mass was lower in the more abundant species in the community and higher in the less abundant ones. These results partly supported the first hypothesis, but not the second. The effect of integration on community structure could be due to higher resource import by the ramets of Alternanthera closer to the dominant species. Integration therefore can increase the initial spread of new clonal plant species into communities and modify the effects of this spread on community structure.     

Adaptation of protein expression by Escherichia coli in the gastrointestinal tract of gnotobiotic mice

The gastrointestinal tract of mammals is inhabited by several hundred bacterial species. While the effects of the gut microbiota upon the host have been widely studied, the microbial response to host factors has only recently attracted attention. In order to investigate the influence of the host on the physiology of gastrointestinal bacteria, a simplified model of host–bacteria interaction was created by associating germfree mice with commensal Escherichia coli . Here we demonstrate the feasibility of analysing the bacterial response to the conditions in the digestive system by a proteomics-based approach. Two-dimensional gel electrophoresis (2D-GE) followed by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) was used to identify bacterial proteins from caecal and faecal samples. In a set of 60 arbitrarily chosen spots of stably and differentially expressed proteins, 50 different bacterial proteins were identified. Their ascribed functions suggest that the host-associated bacteria adapt their metabolism to the conditions in the intestine by utilizing arginine, asparagine and aspartate as well as glucose/galactose, ribose, maltose, glucuronate, galacturonate and gluconate as substrates. Thirteen proteins not previously detected on 2D-gels and 10 proteins with unknown or poorly characterized physiological function were identified, while the existence of three proteins had so far only been inferred from predictions or by homology.