Non-classical proIL-1beta activation during mammary gland infection is pathogen-dependent but caspase-1 independent.

Infection of the mammary gland with live bacteria elicits a pathogen-specific host inflammatory response. To study these host-pathogen interactions wild type mice, NF-kappaB reporter mice as well as caspase-1 and IL-1beta knockout mice were intramammarily challenged with Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The murine mastitis model allowed to compare the kinetics of the induced cytokine protein profiles and their underlying pathways. In vivo and ex vivo imaging showed that E. coli rapidly induced NF-kappaB inflammatory signaling concomitant with high mammary levels of TNF-alpha, IL-1 alpha and MCP-1 as determined by multiplex analysis. In contrast, an equal number of S. aureus bacteria induced a low NF-kappaB activity concomitant with high mammary levels of the classical IL-1beta fragment. These quantitative and qualitative differences in local inflammatory mediators resulted in an earlier neutrophil influx and in a more extensive alveolar damage post-infection with E. coli compared to S. aureus. Western blot analysis revealed that the inactive proIL-1beta precursor was processed into pathogen-specific IL-1beta fragmentation patterns as confirmed with IL-1beta knockout animals. Additionally, caspase-1 knockout animals allowed to investigate whether IL-1beta maturation depended on the conventional inflammasome pathway. The lack of caspase-1 did not prevent extensive proIL-1beta fragmentation by either of S. aureus or E. coli. These non-classical IL-1beta patterns were likely caused by different proteases and suggest a sentinel function of IL-1beta during mammary gland infection. Thus, a key signaling nodule can be defined in the differential host innate immune defense upon E. coli versus S. aureus mammary gland infection, which is independent of caspase-1.

Coupling of drug containing liposomes to microbubbles improves ultrasound triggered drug delivery in mice.

Local extravasation and triggered drug delivery by use of ultrasound and microbubbles is a promising strategy to target drugs to their sites of action. In the past we have developed drug loaded microbubbles by coupling drug containing liposomes to the surface of microbubbles. Until now the advantages of this drug loading strategy have only been demonstrated in vitro. Therefore, in this paper, microbubbles with indocyanine green (ICG) containing liposomes at their surface or a mixture of ICG-liposomes and microbubbles was injected intravenously in mice. Immediately after injection the left hind leg was exposed to 1 MHz ultrasound and the ICG deposition was monitored 1, 4 and 7 days post-treatment by in vivo fluorescence imaging. In mice that received the ICG-liposome loaded microbubbles the local ICG deposition was, at each time point, about 2-fold higher than in mice that received ICG-liposomes mixed with microbubbles. We also showed that the perforations in the blood vessels allow the passage of ICG-liposomes up to 5h after microbubble and ultrasound treatment. An increase in tissue temperature to 41°C was observed in all ultrasound treated mice. However, ultrasound tissue heating was excluded to cause the local ICG deposition. We concluded that coupling of drug containing liposomes to microbubbles may increase ultrasound mediated drug delivery in vivo.

Comparison of in vivo optical systems for bioluminescence and fluorescence imaging.

In vivo optical imaging has become a popular tool in animal laboratories. Currently, many in vivo optical imaging systems are available on the market, which often makes it difficult for research groups to decide which system fits their needs best. In this work we compared different commercially available systems, which can measure both bioluminescent and fluorescent light. The systems were tested for their bioluminescent and fluorescent sensitivity both in vitro and in vivo. The IVIS Lumina II was found to be most sensitive for bioluminescence imaging, with the Photon Imager a close second. Contrary, the Kodak system was, in vitro, the most sensitive system for fluorescence imaging. In vivo, the fluorescence sensitivity of the systems was similar. Finally, we examined the added value of spectral unmixing algorithms for in vivo optical imaging and demonstrated that spectral unmixing resulted in at least a doubling of the in vivo sensitivity. Additionally, spectral unmixing also enabled separate imaging of dyes with overlapping spectra which were, without spectral unmixing, not distinguishable.

Enhancing nucleic acid delivery with ultrasound and microbubbles.

For gene therapy to work in vivo, nucleic acids need to reach the target cells without causing major side effects to the patient. In many cases the gene only has to reach a subset of cells in the body. Therefore, targeted delivery of genes to the desired tissue is a major issue in gene delivery. Many different possibilities of targeted gene delivery have been studied. A relatively novel approach to target nucleic acids and other drugs to specific regions in the body is the use of ultrasound and microbubbles. Microbubbles are gas-filled spheres with a stabilizing lipid, protein, or polymer shell. When these microbubbles enter an ultrasonic field, they start to oscillate. The bubble expansion and compression are inversely related to the pressure phases in the ultrasonic field. When microbubbles are exposed to high-intensity ultrasound they will eventually implode and fragment. This generates shockwaves and microjets which can temporarily permeate cell membranes and blood vessels. Nucleic acids or (non)-viral vectors can extravasate through these pores to gain access to the cell's cytoplasm or the surrounding tissue. The nucleic acids can either be mixed with the microbubbles or loaded on the microbubbles. Nucleic acid-loaded microbubbles can be obtained by coupling nucleic acid-containing particles (i.e., lipoplexes) to the microbubbles. Upon ultrasound-mediated implosion of the microbubbles, the nucleic acid-containing particles will be released and will deliver their nucleic acids in the ultrasound-targeted region.