Mesenchymal stromal cells (MSCs) are currently under investigation for the treatment of inflammatory disorders, including Crohn's disease. MSCs are pluripotent cells with immunosuppressive properties. Recent data suggest that resting MSCs do not have significant immunomodulatory activity, but that the immunosuppressive function of MSCs has to be elicited by interferon-c (IFN-c). In this article, we assessed the effects of IFN-c prestimulation of MSCs (IMSCs) on their immunosuppressive properties in vitro and in vivo. To this end, we pretreated MSCs with IFN-c and assessed their therapeutic effects in dextran sodium sulfate (DSS)-and trinitrobenzene sulfonate (TNBS)-induced colitis in mice. We found that mice treated with IMSCs (but not MSCs) showed a significantly attenuated development of DSS-induced colitis. Furthermore, IMSCs alleviated symptoms of TNBS-induced colitis. IMSCtreated mice displayed an increase in body weight, lower colitis scores, and better survival rates compared with untreated mice. In addition, serum amyloid A protein levels and local proinflammatory cytokine levels in colonic tissues were significantly suppressed after administration of IMSC. We also observed that IMSCs showed greater migration potential than unstimulated MSCs to sites within the inflamed intestine. In conclusion, we show that prestimulation of MSCs with IFN-c enhances their capacity to inhibit Th1 inflammatory responses, resulting in diminished mucosal damage in experimental colitis. These data demonstrate that IFN-c activation of MSCs increases their immunosuppresive capacities and importantly, their therapeutic efficacy in vivo. STEM CELLS 2011;

Prosthetic joint replacement surgery is performed with increasing frequency. Overall the incidence of prosthetic joint infection (PJI) and subsequently prosthesis revision failure is estimated to be between 1 and 3%. Differentiating infection from aseptic mechanical loosening, which is the most common cause of prosthetic failure, is especially important because of different types of therapeutic management. Despite a thorough patient history, physical examination, multiple diagnostic tests and complex algorithms, differentiating PJI from aseptic loosening remains challenging. Among imaging modalities, radiographs are neither sensitive nor specific and cross-sectional imaging techniques, such as computed tomography and magnetic resonance imaging, are limited by hardware-induced artefacts. Radionuclide imaging reflects functional rather than anatomical changes and is not hampered by the presence of a metallic joint prosthesis. As a result scintigraphy is currently the modality of choice in the investigation of suspected PJI. Unfortunately, there is no true consensus about the gold standard technique since there are several drawbacks and limitations inherent to each modality. Bone scintigraphy (BS) is sensitive for identifying the failed joint replacement, but cannot differentiate between infection and aseptic loosening. Combined bone/gallium scintigraphy (BS/GS) offers modest improvement over BS alone for diagnosing PJI. However, due to a number of drawbacks, BS/GS has generally been superseded by other techniques but it still may have a role in neutropenic patients. Radiolabelled leucocyte scintigraphy remains the gold standard technique for diagnosing neutrophil-mediated processes. It seems to be that combined in vitro labelled leucocyte/bone marrow scintigraphy (LS/BMS), with an accuracy of about 90%, is currently the imaging modality of choice for diagnosing PJI. There are, however, significant limitations using in vitro labelled leucocytes and considerable effort has been devoted to developing alternative radiotracers, such as radiolabelled HIGs, liposomes, antigranulocyte antibodies and fragments, as well as more investigational tracers such as radiolabelled antibiotics, antimicrobial peptides, bacteriophages and thymidine kinase. On the other hand, positron emission tomography (PET) is still growing in the field of PJI imaging with radiotracers such as (18)F-fluorodeoxyglucose (FDG), (18)F-FDG white blood cells and (18)F-fluoride. But unfortunately this superb tomographic technique will only receive full acceptance when specific PET uptake patterns can be successfully developed. The emergence of hybrid modality imaging using integrated single photon emission computed tomography (SPECT) and PET with computed tomography (SPECT/CT and PET/CT) may also have a contributing role for more accurate assessment of joint replacement complications, especially combined with new radiotracers such as (68)Ga and (64)Cu. Finally, in searching for infection-specific tracers, currently there is no such diagnostic agent available.

Since human lactoferrin (hLF) binds to bacterial products through its highly positively charged N terminus, we investigated which of the two cationic domains is involved in its bactericidal activity. The results revealed that hLF lacking the first three residues (hLF ؊3N ) was less efficient than hLF in killing of antibiotic-resistant Staphylococcus aureus, Listeria monocytogenes, and Klebsiella pneumoniae. Both hLF preparations failed to kill Escherichia coli O54. In addition, hLF ؊3N was less effective than hLF in reducing the number of viable bacteria in mice infected with antibiotic-resistant S. aureus and K. pneumoniae. Studies with synthetic peptides corresponding to the first 11 N-terminal amino acids, designated hLF(1-11), and fragments thereof demonstrated that peptides lacking the first three N-terminal residues are less effective than hLF(1-11) in killing of bacteria. Furthermore, a peptide corresponding to residues 21 to 31, which comprises the second cationic domain, was less effective than hLF(1-11) in killing of bacteria in vitro and in mice having an infection with antibioticresistant S. aureus or K. pneumoniae. Using fluorescent probes, we found that bactericidal hLF peptides, but not nonbactericidal peptides, caused an increase of the membrane permeability. In addition, hLF killed the various bacteria, most probably by inducing intracellular changes in these bacteria without affecting the membrane permeability. Together, hLF and peptides derived from its N terminus are highly effective against infections with antibiotic-resistant S. aureus and K. pneumoniae, and the first two arginines play an essential role in this activity.

The aim of this study was to select technetium-99m labelled peptides that can discriminate between bacterial infections and sterile inflammations. For this purpose, we first assessed the binding of various 99mTc-labelled natural or synthetic peptides, which are based on the sequence of the human antimicrobial peptide ubiquicidin (UBI) or human lactoferrin (hLF), to bacteria and to leucocytes in vitro. In order to select peptides that preferentially bind to bacteria over host cells, radiolabelled peptides were injected into mice intraperitoneally infected with Klebsiella pneumoniae (K. pneumoniae) and the amount of radioactivity associated with the bacteria and with the leucocytes was quantitated. The next phase focussed on discrimination between bacterial infections and sterile inflammatory processes using 99mTc-labelled peptides in mice intramuscularly infected with various bacteria (e.g. multi-drug-resistant Staphylococcus aureus) and in animals that had been injected with lipopolysaccharides (LPS) of bacterial origin to create a sterile inflammatory process. Also, we studied the distribution of 99mTc-labelled UBI 29-41 and UBI 18-35 in rabbits having an experimental thigh muscle infection with K. pneumoniae and in rabbits injected with LPS. Based on the results of our in vitro and in vivo binding assays, two peptides, i.e. UBI 29-41 and UBI 18-35, were selected as possible candidates for infection imaging. The radiolabelled peptides can detect infections with both gram-positive and gram-negative bacteria in mice as early as 5-30 min after injection, with a target-to-non-target (T/NT) ratio between 2 and 3; maximum T/NT ratios were seen within 1 h after injection. In rabbits, high T/NT ratios (>5) for 99mTc-labelled UBI 29-41 were observed from 1 h after injection. No accumulation of the selected 99mTc-labelled UBI-derived peptides was observed in thighs of mice and rabbits previously injected with LPS. Scintigraphic investigation into the biodistribution of 99mTc-labelled UBI peptides revealed that these peptides were rapidly removed from the circulation by renal excretion. Similar data were observed for 99mTc-labelled defensin 1-3. Our data for 99mTc-labelled hLF and related peptides indicate that these compounds are less favourable for infection detection. Taken together, 99mTc-labelled UBI 18-35 and UBI 29-41 enable discrimination between bacterial infections and sterile inflammatory processes in both mice and rabbits. Based on their characteristics, we consider these peptides the candidates of preference for detection of bacterial infections in man.

The limited capacity of current bioreactors has led the biopharmaceutical industry to investigate alternative protein expression systems. The milk of transgenic cattle may provide an attractive vehicle for large-scale production of biopharmaceuticals, but there have been no reports on the characteristics of such recombinant proteins. Here we describe the production of recombinant human lactoferrin (rhLF), an iron-binding glycoprotein involved in innate host defense, at gram per liter concentrations in bovine milk. Natural hLF from human milk and rhLF had identical iron-binding and -release properties. Although natural hLF and rhLF underwent differential N-linked glycosylation, they were equally effective in three different in vivo infection models employing immunocompetent and leukocytopenic mice, and showed similar localization at sites of infection. Taken together, the results illustrate the potential of transgenic cattle in the large-scale production of biopharmaceuticals.