Identification of a Live Attenuated Vaccine Candidate for Tularemia Prophylaxis

Francisella tularensis is the causative agent of a fatal human disease, tularemia. F. tularensis was used in bioweapon programs in the past and is now classified as a category A select agent owing to its possible use in bioterror attacks. Despite over a century since its discovery, an effective vaccine is yet to be developed. In this study four transposon insertion mutants of F. tularensis live vaccine strain (LVS) in Na/H antiporter (FTL_0304), aromatic amino acid transporter (FTL_0291), outer membrane protein A (OmpA)-like family protein (FTL_0325) and a conserved hypothetical membrane protein gene (FTL_0057) were evaluated for their attenuation and protective efficacy against F. tularensis SchuS4 strain. All four mutants were 100–1000 fold attenuated for virulence in mice than parental F. tularensis. Except for the FTL_0304, single intranasal immunization with the other three mutants provided 100% protection in BALB/c mice against intranasal challenge with virulent F. tularensis SchuS4. Differences in the protective ability of the FTL_0325 and FTL_0304 mutant which failed to provide protection against SchuS4 were investigated further. The results indicated that an early pro-inflammatory response and persistence in host tissues established a protective immunity against F. tularensis SchuS4 in the FTL_0325 immunized mice. No differences were observed in the levels of serum IgG antibodies amongst the two vaccinated groups. Recall response studies demonstrated that splenocytes from the FTL_0325 mutant immunized mice induced significantly higher levels of IFN-γ and IL-17 cytokines than the FTL_0304 immunized counterparts indicating development of an effective memory response. Collectively, this study demonstrates that persistence of the vaccine strain together with its ability to induce an early pro-inflammatory innate immune response and strong memory responses can discriminate between successful and failed vaccinations against tularemia. This study describes a live attenuated vaccine which may prove to be an ideal vaccine candidate for prevention of respiratory tularemia.     

Triggering of cultured neoplastic mast cells by antibodies to the receptor for IgE.

Cell surface receptors for IgE were isolated from detergent lysates of iodinated, IgE-saturated, rat basophilic leukemia cells by precipitation with anti-IgE antibodies followed by chromatography at acid pH. The isolated material showed a single 125I-band (m.w. approximately 58,000) on gel electrophoresis in sodium dodecyl sulfate and was used to immunize a rabbit. The resulting anti-serum was reacted with lysates of surface iodinated mouse or rat tumor mast cells. Analysis of the precipitates on (10%) gel electrophoresis revealed one major peak comprising greater than 80% of the detectable counts and having an estimated m.w. of approximately 58,000. The antiserum reacted with detergent-solubilized and cell-bound receptors in the presence or absence of excess IgE; it also inhibited the binding of 125I-IgE. Cultured mouse mastocytoma cells never exposed to IgE released 3H-serotonin when incubated with F(ab')2, but not Fab' fragments of the antiserum, which had been rigorously freed of IgE and anti-IgE. The release was inhibited in the presence of excess IgE, was Ca++ dependent, and equaled 80% of the maximum obtained with IgE and anti-IgE. We conclude that aggregation of the receptors for IgE provides the critical signals for cell activation.     

Direct,Differential Effects of Tamoxifen, 4-Hydroxytamoxifen,and Raloxifene on Cardiac Myocyte Contractility and Calcium Handling

Tamoxifen (Tam), a selective estrogen receptor modulator, is in wide clinical use for the treatment and prevention of breast cancer. High Tam doses have been used for treatment of gliomas and cancers with multiple drug resistance, but long QT Syndrome is a side effect. Tam is also used experimentally in mice for inducible gene knockout in numerous tissues, including heart; however, the potential direct effects of Tam on cardiac myocyte mechanical function are not known. The goal of this study was to determine the direct, acute effects of Tam, its active metabolite 4-hydroxytamoxifen (4OHT), and related drug raloxifene (Ral) on isolated rat cardiac myocyte mechanical function and calcium handling. Tam decreased contraction amplitude, slowed relaxation, and decreased Ca²⁺ transient amplitude. Effects were primarily observed at 5 and 10 μM Tam, which is relevant for high dose Tam treatment in cancer patients as well as Tam-mediated gene excision in mice. Myocytes treated with 4OHT responded similarly to Tam-treated cells with regard to both contractility and calcium handling, suggesting an estrogen-receptor independent mechanism is responsible for the effects. In contrast, Ral increased contraction and Ca²⁺ transient amplitudes. At 10 μM, all drugs had a time-dependent effect to abolish cellular contraction. In conclusion, Tam, 4OHT, and Ral adversely and differentially alter cardiac myocyte contractility and Ca²⁺ handling. These findings have important implications for understanding the Tam-induced cardiomyopathy in gene excision studies and may be important for understanding effects on cardiac performance in patients undergoing high-dose Tam therapy.     

Specific and Redundant Functions of Retinoid X Receptor/Retinoic Acid Receptor Heterodimers in Differentiation, Proliferation, and Apoptosis of F9 Embryonal Carcinoma Cells

We have generated F9 murine embryonal carcinoma cells in which either the retinoid X receptor (RXR)α and retinoic acid receptor (RAR)α genes or the RXRα and RARγ genes are knocked out, and compared their phenotypes with those of wild-type (WT), RXRα^−/−, RARα^−/−, and RARγ^−/− cells. RXRα^−/−/ RARα^−/− cells were resistant to retinoic acid treatment for the induction of primitive and parietal endodermal differentiation, as well as for antiproliferative and apoptotic responses, whereas they could differentiate into visceral endodermlike cells, as previously observed for RXRα^−/− cells. In contrast, RXRα^−/−/RARγ^−/− cells were defective for all three types of differentiation, as well as antiproliferative and apoptotic responses, indicating that RXRα and RARγ represent an essential receptor pair for these responses. Taken together with results obtained by treatment of WT and mutant F9 cells with RAR isotype– and panRXR-selective retinoids, our observations support the conclusion that RXR/ RAR heterodimers are the functional units mediating the retinoid signal in vivo. Our results also indicate that the various heterodimers can exert both specific and redundant functions in differentiation, proliferation, and apoptosis. We also show that the functional redundancy exhibited between RXR isotypes and between RAR isotypes in cellular processes can be artifactually generated by gene knockouts. The present approach for multiple gene targeting should allow inactivation of any set of genes in a given cell.     

Pattern of tree species diversity in riparian forest fragments of different widths (SE Brazil)

Width is an essential element of the spatial configuration of riparian forests and may be fundamental in determining their corridor function. In the present study we tested the effect of forest width on floristic structure (tree species composition and diversity) in 15 fragments of riparian forest in an agricultural fragmented landscape of SE Brazil. All these fragments were chosen in a geomorphological homogeneous river reach under similar soil, topographic and human disturbance conditions in order to minimize the influence of these factors. The forest widths considered ranged from 30 to 650 m. The results showed that total species richness and climax species richness were significantly greater when we consider larger fragments, as has been observed in other studies. Nevertheless, species diversity and evenness were not significantly correlated with forest width. The analysis of species composition showed that the narrowest fragments were characterized by species well adapted to temporary flood conditions, while medium and wide fragments showed a composition typical of drier upland areas. Therefore, the effect of forest width on floristic structure appears to be more strongly linked to the effect of river floods in the case of the fragments studied. The existence in riparian corridors of a drier forest, in general richer and more diversified than the annually flooded forest, seems to favor the maintenance of regional species diversity in fragmented landscapes.     

VARIABILITY OF CELL WALL STRUCTURE AND HYDROCARBON TYPE IN DIFFERENT STRAINS OF BOTRYOCOCCUS BRAUNII1

Different samples of Botryococcus braunii Kütz., freshly collected from nature or laboratory-grown from culture collection strains, were studied by electron microscopy and their hydrocarbon content analyzed. Although the general internal structure of the cells was rather constant, the organization of the outer walls forming the hydrocarbon-rich matrix of the colonies differed greatly from one sample to another. In the majority of cultivated strains, the colonies were rather small, the different successive external walls remained distinct and all strains contained dienic or trienic hydrocarbons. In contrast, most of the collected samples possessed large colonies with a rather compact matrix formed by the hydrocarbon-rich part of the successive closely appressed external wall layers. These samples contained polyunsaturated hydrocarbons, i.e. botryococcenes. Well defined cell caps which sheared off the cells were observed only in those strains with a compact matrix. The Austin strain and some collected samples, however, were intermediate with rather small colonies, dense matrix, definite cell caps and dienic hydrocarbons. Thus, the hydrocarbon composition did not correlate directly with the variations in wall structure; however, the occurence of dienic and botryococcene-like hydrocarbons together in one strain was never observed, although analyzed at various stages of growth. Thus, the existence of distinct strains of Botryococcus braunii, some synthesizing dienes, others botryococcenes, appears highly probable.