Intranasal administration of poly(I:C) and LPS in BALB/c mice induces airway hyperresponsiveness and inflammation via different pathways

Background

Bacterial and viral infections are known to promote airway hyperresponsiveness (AHR) in asthmatic patients. The mechanism behind this reaction is poorly understood, but pattern recognizing Toll-like receptors (TLRs) have recently been suggested to play a role.

Materials and Methods

To explore the relation between infection-induced airway inflammation and the development of AHR, poly(I:C) activating TLR3 and LPS triggering TLR4, were chosen to represent viral and bacterial induced interactions, respectively. Female BALB/c or MyD88-deficient C57BL/6 mice were treated intranasally with either poly(I:C), LPS or PBS (vehicle for the control group), once a day, during 4 consecutive days.

Results

When methacholine challenge was performed on day 5, BALB/c mice responded with an increase in airway resistance. The maximal resistance was higher in the poly(I:C) and LPS treated groups than among the controls, indicating development of AHR in response to repeated TLR activation. The proportion of lymphocytes in broncheoalveolar lavage fluid (BALF) increased after poly(I:C) treatment whereas LPS enhanced the amount of neutrophils. A similar cellular pattern was seen in lung tissue. Analysis of 21 inflammatory mediators in BALF revealed that the TLR response was receptor-specific. MyD88-deficient C57BL/6 mice responded to poly (I:C) with an influx of lymphocytes, whereas LPS caused no inflammation.

Conclusion

In vivo activation of TLR3 and TLR4 in BALB/c mice both caused AHR in conjunction with a local inflammatory reaction. The AHR appeared to be identical regardless of which TLR that was activated, whereas the inflammation exhibited a receptor specific profile in terms of both recruited cells and inflammatory mediators. The inflammatory response caused by LPS appeared to be dependent on MyD88 pathway. Altogether the presented data indicate that the development of AHR and the induction of local inflammation might be the result of two parallel events, rather than one leading to another.     

Protective Vaccination against Infectious Bursal Disease Virus with Whole Recombinant Kluyveromyces lactis Yeast Expressing the Viral VP2 Subunit

Here we report on vaccination approaches against infectious bursal disease (IBD) of poultry that were performed with complete yeast of the species Kluyveromyces lactis (K. lactis). Employing a genetic system that enables the rapid production of stably transfected recombinant K. lactis, we generated yeast strains that expressed defined quantities of the virus capsid forming protein VP2 of infectious bursal disease virus (IBDV). Both, subcutaneous as well as oral vaccination regiments with the heat-inactivated but otherwise untreated yeast induced IBDV-neutralizing antibodies in mice and chickens. A full protection against a subsequent IBDV infection was achieved by subcutaneous inoculation of only milligram amounts of yeast per chicken. Oral vaccination also generated protection: while mortality was observed in control animals after virus challenge, none of the vaccinees died and ca. one-tenth were protected as indicated by the absence of lesions in the bursa of Fabricius. Recombinant K. lactis was thus indicated as a potent tool for the induction of a protective immune response by different applications. Subcutaneously applied K. lactis that expresses the IBDV VP2 was shown to function as an efficacious anti-IBD subunit vaccine.     

Measurement and modelling of ABA signalling in potato (Solanum tuberosum L.) during partial root-zone drying

The objective of this study was to develop a simple mechanistic model to predict the magnitude of ABA signalling ([X-ABA]) of potatoes (Solanum tuberosum L.) exposed to partial root-zone drying (PRD). Potatoes were grown in pots in a glasshouse with the roots split equally between two soil columns. At tuber initiation stage, plants were subjected to three irrigation treatments: (1) both soil columns were fully irrigated (FI) daily to a volumetric soil water content (θ) of 18.0%; (2) PRD, in which one soil column was irrigated daily to 18.0% while the other was allowed to dry, and the irrigation was shifted between columns when the θ of the drying soil column had decreased to 7–8%; (3) non-irrigation (NI), where irrigation was withheld after onset of treatments and lasted for 5 days until θ had decreased to 7%. In the PRD plants, the fraction of soil water extraction (FSWE) by the dry roots declined exponentially with declining soil water potential (Ψ_soil-dry); however, after shifting of irrigation, the previously dried roots immediately recovered the full capacity of water uptake. During the first PRD drying cycle, FI plants had the highest stomatal conductance (g_s), and followed by PRD plants and NI plants had the lowest g_s. Photosynthesis (A) was similar for FI and PRD plants, and was significantly lower for the NI plants only on 3–4 days after treatment. In the NI plants, a linear relationship between Ψ_soil and [X-ABA] was obtained. Based on these relationships, a simple model predicting [X-ABA] in the PRD plants ([X-ABA]_PRD) was developed. Assuming that a constant [X-ABA] of 115 nM (similar to that found in the FI plants) originated from the wet roots; the simulation results indicated that irrigation should be shifted between the two sides when Ψ_soil-dry had decreased to −80 kPa, and [X-ABA]_PRD had reached a peak of ca. 150 nM. However, the [X-ABA]_PRD predicted by the model was significantly lower than the measured value; whilst a simple average of [X-ABA] from the wet and the dry soil columns based on the [X-ABA]–Ψ_soil relationship better predicted [X-ABA]_PRD.     

Correlation of beta-camera imaging and immunohistochemistry in radioimmunotherapy using90Y-labeled monoclonal antibodies in ovarian cancer animal models

Tumor stroma contains much fibrin and monoclonal antifibrin antibody targeting is possible in tumors. In this study, nude mouse human ovarian carcinoma xenograft specimens were investigated after treatment with⁹⁰Y-labeled monoclonal antifibrin antibody Fab fragment or with⁹⁰Y-labeled OC125-monoclonal antibody F(ab′)₂ fragments. The mice received the radioimmunotherapy activity either intratumorally, intraperitoneally, or intravenously. Beta-camera imaging (BCI) is a novel device for studying activity distribution in tissue specimens and, together with immunohistochemistry (IHC) with OC125, antifibrin, anticarcinoembryonic antigen, anti-cytokeratin, and anti-placental alkaline phosphatase antibodies, was used for correlation of activity distribution of tissue specimens. These results were in concordance: Antigen distribution measured with IHC and radioactivity distribution were similar with the same antibodies, antifibrin, and OC125: However, these antigens demonstrated rather different distribution. Tissue studies revealed that activity was concentrated also in the necrotic tumor tissue, indicating that cell death was also caused by radiation. Differences in the tumor cell morphology were observed using different routes of administration. With BCI, it is possible to quantitate activities in frozen sections (microdosimetry), and these results were in concordance with absolute activities as measured by tissue sampling and well-counting. Three-dimensional reconstruction of tissue slices combined with radioactivity distribution measured with BCI allows estimation of total absorbed radiation dose in tumor after an appropriate dose planning.     

Biochemical and ultrastructural studies of the C-type lectin bovine conglutinin

The aim of this study was to correlate the supramolecular organization of conglutinin (BK) with its primary and tertiary structure and to gain more knowledge of functionally important regions of the molecule. BK analyzed by SDS-PAGE under standard reducing conditions (40 mM DTT) showed a major band at 43 kDa and weaker bands at 86 and 180 kDa. In contrast, reduction with 6-50 mM L-cysteine resulted in 37-kDa subunits indicating the presence of intrachain disulfide bonds within this subunit. Hydroxylamine treatment indicated presence of ester bonds in the 86- and 180-kDa subunits. Collagenase digestion and SDS-PAGE under reducing and nonreducing conditions resulted in bands of 20 and 15 kDa, respectively, indicating the presence of intrachain, rather than interchain, disulfide bonds in the carboxy terminus. Deglycosylation and glycan differentiation analysis of BK revealed the presence of O-linked glycans of GalNAc and alpha (2-3) linked sialic acid type, whereas no N-linked glycans were demonstrated. Binding experiments with GlcNAc-gold suggested that multivalency is required for carbohydrate binding to BK. Electron microscopy showed mostly tetramers, 96 nm in diameter, but also mono-, di-, and trimers were seen. The tetramers consisted of 40-nm strands, each with a peripheral globular head composed of subunits and connected to a common central lobe built from four ring-formed structures. The strands occasionally showed two bends, one close to the central lobe and another 25 nm from the lobe. These bends most likely correspond to the interrupted Gly-Xaa-Yaa repeats at residues 38 and 123.     

Physiological responses of potato (Solanum tuberosum L.) to partial root-zone drying: ABA signalling, leaf gas exchange, and water use efficiency

The physiological responses of potato (Solanum tuberosum L. cv. Folva) to partial root-zone drying (PRD) were investigated in potted plants in a greenhouse (GH) and in plants grown in the field under an automatic rain-out-shelter. In the GH, irrigation was applied daily to the whole root system (FI), or to one-half of the root system while the other half was dried, for 9 d. In the field, the plants were drip irrigated either to the whole root system near field capacity (FI) or using 70% water of FI to one side of the roots, and shifted to the other side every 5-10 d (PRD). PRD plants had a similar midday leaf water potential to that of FI, whereas in the GH their root water potential (Psi(r)) was significantly lowered after 5 d. Stomatal conductance (g(s)) was more sensitive to PRD than photosynthesis (A) particularly in the field, leading to greater intrinsic water use efficiency (WUE) (i.e. A/g(s)) in PRD than in FI plants on several days. In PRD, the xylem sap abscisic acid concentration ([ABA](xylem)) increased exponentially with decreasing Psi(r); and the relative [ABA](xylem) (PRD/FI) increased exponentially as the fraction of transpirable soil water (FTSW) in the drying side decreased. In the field, the leaf area index was slightly less in PRD than in FI treatment, while tuber biomass was similar for the two treatments. Compared with FI, PRD treatment saved 30% water and increased crop water use efficiency (WUE) by 59%. Restrictions on leaf area expansion and g(s) by PRD-induced ABA signals might have contributed to reduced water use and increased WUE.     

Varietal differences of quinoa’s tolerance to saline conditions

Aims

This study aimed to assess varietal differences of quinoa’s tolerance to salinity and to investigate physiological mechanisms conferring these differences.

Methods

Production of biomass in fourteen varieties grown under saline conditions was analysed in a pot experiment. For two contrasting varieties, the Danish variety Titicaca and the Bolivian variety Utusaya gas exchange, chlorophyll content index (CCI), fluorescence and ion relations were studied.

Results

Responses to salinity differed greatly among the varieties; least affected were two varieties from the Bolivian altiplano and a variety from Peru. Titicaca and Utusaya both had substantially increased K+ concentrations in the leaf sap. But, Utusaya was much more efficient in restricting xylem Na+ loading. Xylem Na+ and K+ loading were found to be uncoupled. Utusaya maintained a relatively high stomatal conductance resulting in an only 25% NaCl-induced reduction in net CO₂ assimilation compared to a 67% reduction in salt treated Titicaca plants. Maximum photochemical efficiency of PSII was not affected by salinity.

Conclusion

In addition to maintaining high gas exchange, tolerant varieties better control xylem Na+ loading. To what extent this control is related to radial root Na+ uptake or to the activity of Na+/H+-exchangers at the xylem parenchyma boundary remains to be studied.