Caspase-1 activation by Salmonella

Salmonella is an interesting example of how the selective pressure of host environments has led to the evolution of sophisticated bacterial virulence mechanisms. This microbe exploits the first-line of defence, the macrophage, as a crucial tool in the initiation of disease. After invasion of intestinal macrophages, a virulence protein secreted by Salmonella specifically induces apoptotic cell death by activating the cysteine protease caspase-1. The pro-apoptotic capability is necessary for successful pathogenesis. The study of mechanisms by which Salmonella induces programmed cell death offers new insights into how pathogens cause disease and into general mechanisms of activation of the innate immune system.     

Effect of access to a running wheel on behavior of C57BL/6J mice.

BACKGROUND AND PURPOSE: To evaluate how and when mice run on a running wheel and how ad libitum access to the wheel affect behavior, feed intake, and weight gain. METHODS: Seventeen 2-month-old C57BL/6J mice had access to the wheel, whereas 19 control mice did not. After 3 to 6.5 weeks, behavior was video-recorded over 24 h for each mouse. RESULTS: Experimental mice ran an average 2 km/24 h in 114 min. Highest running activity took place at the onset of darkness. Experimental mice spent 22 min more feeding on the cage floor than did control mice. These times were deducted from those for all other behaviors: 74 min from resting time, 39 min from climbing and feeding on the cage lid, 14 min from locomotion on the cage floor, and 10 min from grooming. In relative figures, deduction from sleeping time was only 9%, whereas climbing time was halved. CONCLUSIONS: Climbing on the cage lid has a similar circadian rhythm as does wheel running and high-energy expenditure. Because experimental mice climbed less, their weight gain and feed intake were similar to those of control mice. Thus, wheel running can substitute for other forms of energy-consuming behaviors and vice versa.     

Function and regulation of Alx4 in limb development: complex genetic interactions with Gli3 and Shh

The role of the aristaless-related homeobox gene Alx4 in antero-posterior (AP-) patterning of the developing vertebrate limb has remained somewhat elusive. Polydactyly of Alx4 mutant mice is known to be accompanied by ectopic anterior expression of genes like Shh, Fgf4 and 5'Hoxd. We reported previously that polydactyly in Alx4 mutant mice requires SHH signaling, but we now show that in early Alx4-/- limb buds the anterior ectopic expression of Fgf4 and Hoxd13, and therefore disruption of AP-patterning, occurs independently of SHH signaling. To better understand how Alx4 functions in the pathways that regulate AP-patterning, we also studied genomic regulatory sequences that are capable of directing expression of a reporter gene in a pattern corresponding to endogenous Alx4 expression in anterior limb bud mesenchyme. We observed, as expected for authentic Alx4 expression, expansion of reporter construct expression in a Shh-/- background. Total lack of reporter expression in a Gli3-/- background confirms the existence of Gli3-dependent and -independent Alx4 expression in the limb bud. Apparently, these two modules of Alx4 expression are linked to dissimilar functions.     

Topical superantigen exposure induces epidermal accumulation of CD8+ T cells, a mixed Th1/Th2-type dermatitis and vigorous production of IgE antibodies in the murine model of atopic dermatitis

Patients with atopic dermatitis (AD) have repeated cutaneous exposure to both environmental allergens and superantigen-producing strains of Staphylococcus aureus. We used a murine model of AD to investigate the role of staphylococcal enterotoxin B (SEB) in the modulation of allergen-induced skin inflammation. Mice were topically exposed to SEB, OVA, a combination of OVA and SEB (OVA/SEB), or PBS. Topical SEB and OVA/SEB exposure induced epidermal accumulation of CD8+ T cells and TCRVbeta8+ cells in contrast to OVA application, which induced a mainly dermal infiltration of CD4+ cells. SEB and OVA/SEB exposure elicited a mixed Th1/Th2-associated cytokine and chemokine expression profile within the skin. Restimulation of lymph node cells from OVA- and OVA/SEB-exposed mice with OVA elicited strong production of IL-13 protein, whereas substantial amounts of IFN-gamma protein were detected after SEB stimulation of cells derived from SEB- or OVA/SEB-exposed mice. Topical SEB treatment elicited vigorous production of SEB-specific IgE and IgG2a Abs and significantly increased the production of OVA-specific IgE and IgG2a Abs. The present study shows that topical exposure to SEB provokes epidermal accumulation of CD8+ T cells, a mixed Th2/Th1 type dermatitis and vigorous production of specific IgE and IgG2a Abs, which can be related to the chronic phase of atopic skin inflammation.     

Preservation of dried liposomes in the presence of sugar and phosphate

It has been well established that sugars can be used to stabilize liposomes during drying by a mechanism that involves the formation of a glassy state by the sugars as well as by a direct interaction between the sugar and the phospholipid head groups. We have investigated the protective effect of phosphate on solute retention and storage stability of egg phosphatidylcholine (egg PC) liposomes that were dried (air-dried and freeze-dried) in the presence of sugars and phosphate. The protective effect of phosphate was tested using both glucose (low T(g)) and sucrose (high T(g)) by measuring leakage of carboxyfluorescein (CF), which was incorporated inside the vesicles. Liposomes that were dried with glucose or phosphate alone showed complete leakage after rehydration. However, approximately 30% CF-retention was obtained using mixtures of phosphate and glucose. Approximately 75% CF-retention was observed with liposomes that were dried with sucrose. The solute retention further increased to 85% using mixtures of phosphate and sucrose. The pH of the phosphate buffer prior to drying was found to have a strong effect on the solute retention. Fourier transform infrared spectroscopy studies showed that phosphate and sugars form a strong hydrogen bonding network, which dramatically increased the T(g). The HPO(4)(2-) form of phosphate was found to interact stronger with sugars than the H(2)PO(4)(-) form. The increased solute retention of liposomes dried in the sugar phosphate mixtures did not coincide with improved storage stability. At temperatures below 60 degrees C the rate of solute-leakage was found to be strikingly higher in the presence of phosphate, indicating that phosphate impairs storage stability of dried liposomes.     

Introduction of lanthanide(III) chelates to oligopeptides on solid phase

The synthesis of oligopeptide building blocks for the introduction of nonluminescent and luminescent lanthanide(III) chelates to the oligopeptide structure on the solid phase is described. The oligopeptide conjugates synthesized were used in DELFIA-based receptor binding assay (motilin) as well as in LANCE time-resolved fluorescence quenching assay (caspase-3).     

Attenuated expression of tenascin-c in ovalbumin-challenged STAT4-/- mice

Background

Asthma leads to structural changes in the airways, including the modification of extracellular matrix proteins such as tenascin-C. The role of tenascin-C is unclear, but it might act as an early initiator of airway wall remodelling, as its expression is increased in the mouse and human airways during allergic inflammation. In this study, we examined whether Th1 or Th2 cells are important regulators of tenascin-C in experimental allergic asthma utilizing mice with impaired Th1 (STAT4-/-) or Th2 (STAT6-/-) immunity.

Methods

Balb/c wildtype (WT), STAT4-/- and STAT6-/- mice were sensitized with intraperitoneally injected ovalbumin (OVA) followed by OVA or PBS airway challenge. Airway hyperreactivity (AHR) was measured and samples were collected. Real time PCR and immunohistochemistry were used to study cytokines and differences in the expression of tenascin-C. Tenascin-C expression was measured in human fibroblasts after treatment with TNF-α and IFN-γ in vitro.

Results

OVA-challenged WT mice showed allergic inflammation and AHR in the airways along with increased expression of TNF-α, IFN-γ, IL-4 and tenascin-C in the lungs. OVA-challenged STAT4-/- mice exhibited elevated AHR and pulmonary eosinophilia. The mRNA expression of TNF-α and IFN-γ was low, but the expression of IL-4 was significantly elevated in these mice. OVA-challenged STAT6-/- mice had neither AHR nor pulmonary eosinophilia, but had increased expression of mRNA for TNF-α, IFN-γ and IL-4. The expression of tenascin-C in the lungs of OVA-challenged STAT4-/- mice was weaker than in those of OVA-challenged WT and STAT6-/- mice suggesting that TNF-α and IFN-γ may regulate tenascin-C expression in vivo. The stimulation of human fibroblasts with TNF-α and IFN-γ induced the expression of tenascin-C confirming our in vivo findings.

Conclusions

Expression of tenascin-C is significantly attenuated in the airways of STAT4-/- mice, which may be due to the impaired secretion of TNF-α and IFN-γ in these mice.     

Nutrient concentration in shape Sphagna at increased N-deposition rates and raised atmospheric CO2 concentrations

Sphagnum fuscum, S. magellanicum, S. angustifolium and S. warnstorfii were treated with N deposition rates (0, 10, 30 and 100 kg ha^-1 a^-1) and with four atmospheric CO₂ concentrations (350, 700, 1000 and 2000 ppm) in greenhouse for 71–120 days. Thereafter, concentrations of total N, P, K, Ca and Mg in the capitulae of the Sphagna were determined. The response of each species to N deposition was related to ecological differences. With increasing N deposition treatments, moss N concentrations increased and higher N:P-ratios were found, the increase being especially clear at the highest N load. Sphagnum fuscum, which occupies ombrotrophic habitats, was the most affected by the increased nitrogen load and as a consequence the other elements were decreased. Oligotrophic S. magellanicum, wide nutrient status tolerant S. angustifolium and meso-eutrophic S. warnstorfii tolerated better increased N deposition, though there were increased concentrations of Ca and Mg in S. warnstorfii and Mg in S. magellanicum. Nitrogen and P concentrations decreased with raised CO₂ concentrations, except for S. magellanicum. This seems to be the first time this kind of response in nutrient concentrations to enhanced CO₂ concentration has been shown to exist in bryophytes. The concentration of K clearly decreased in S. fuscum as did the concentration of Mg in the other Sphagna with increasing CO₂. Sphagnum angustifolium and S. magellanicum, which are the less specialized species, were the least affected by the CO₂ treatments.