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The C3 toxins from Clostridium botulinum (C3bot) and Clostridium limosum (C3lim) as well as C3-derived fusion proteins are selectively taken up into the cytosol of monocytes/macrophages where the C3-catalyzed ADP-ribosylation of Rho results in inhibition of Rho-signalling and characteristic morphological changes. Since the fusion toxin C2IN-C3lim was efficiently taken up into and inhibited proliferation of murine macrophage-like RAW 264.7 cells, its effects on RAW 264.7-derived osteoclasts were investigated. C2IN-C3lim was taken up into differentiated osteoclasts and decreased their resorption activity. In undifferentiated RAW 264.7 cells, C2IN-C3lim-treatment significantly decreased their differentiation into osteoclasts as determined by counting the multi-nucleated, TRAP-positive cells. This inhibitory effect was concentration- and time-dependent and most efficient when C2IN-C3lim was applied in the early stage of osteoclast-formation. A single-dose application of C2IN-C3lim at day 0 and its subsequent removal at day 1 reduced the number of osteoclasts in a comparable manner while C2IN-C3lim-application at later time points did not reduce the number of osteoclasts to a comparable degree. Control experiments with an enzymatically inactive C3 protein revealed that the ADP-ribosylation of Rho was essential for the observed effects. In conclusion, the results indicate that Rho-activity is crucial during the early phase of osteoclast-differentiation. Other bone cell types such as pre-osteoblastic cells were not affected by C2IN-C3lim. Due to their cell-type selective and specific mode of action, C3 proteins and C3-fusions might be valuable tools for targeted pharmacological manipulation of osteoclast formation and activity, which could lead to development of novel therapeutic strategies against osteoclast-associated diseases.

Background: The C3bot1 protein (~23 kDa) from Clostridium botulinum ADP-ribosylates and thereby inactivates Rho. C3bot1 is selectively taken up into the cytosol of monocytes/macrophages but not of other cell types such as epithelial cells or fibroblasts. Most likely, the internalization occurs by a specific endocytotic pathway via acidified endosomes., Methodology/principal Findings: Here, we tested whether enzymatic inactive C3bot1E174Q serves as a macrophage-selective transport system for delivery of enzymatic active proteins into the cytosol of such cells. Having confirmed that C3bot1E174Q does not induce macrophage activation, we used the actin ADP-ribosylating C2I (∼50 kDa) from Clostridium botulinum as a reporter enzyme for C3bot1E174Q-mediated delivery into macrophages. The recombinant C3bot1E174Q-C2I fusion toxin was cloned and expressed as GST-protein in Escherichia coli. Purified C3bot1E174Q-C2I was recognized by antibodies against C2I and C3bot and showed C2I-specific enzyme activity in vitro. When applied to cultured cells C3bot1E174Q-C2I ADP-ribosylated actin in the cytosol of macrophages including J774A.1 and RAW264.7 cell lines as well as primary cultured human macrophages but not of epithelial cells. Together with confocal fluorescence microscopy experiments, the biochemical data indicate the selective uptake of a recombinant C3-fusion toxin into the cytosol of macrophages., Conclusions/significance: In summary, we demonstrated that C3bot1E174Q can be used as a delivery system for fast, selective and specific transport of enzymes into the cytosol of living macrophages. Therefore, C3-based fusion toxins can represent valuable molecular tools in experimental macrophage pharmacology and cell biology as well as attractive candidates to develop new therapeutic approaches against macrophage-associated diseases.

The antibody-stimulating activities and side effects of commercially available adjuvants were compared in BALB/c mice immunised with the immunosuppressive (ISU) peptide of HIV I. Clinical and pathological-histological parameters as well as behavioural changes, were used to assess the distress and pain caused to the animals. Complete Freund's Adjuvant (FAk) was used as the positive control and PBS as the negative control. The oil-based adjuvants Montanide ISA 51(R) (M 51), Specol(R), and Hunter's TiterMax Gold(R) (HTMG) were used with the ISU-peptide conjugated to KLH. The water-soluble Gerbu Adjuvant(R) was administered together with the antigen KLH conjugate and also with the ISU-peptide conjugated to cholera toxin B subunit (ChTxB). The Dutch "Code of Practice" was used as a guideline for all immunisations. No changes in animal activity or behaviour was observed in any of the groups. All the oil-based adjuvants gave rise to swellings and encapsulations, which were most pronounced in the HTMG-group. Although body weight increased throughout the study, no increase was seen in any adjuvant group for a short period after each booster immunisation, nor after the first immunisation in the HTMG group. FAk induced a light fever after all immunisations. FAk, Specol and M 51 as well as Gerbu-ChTxB induced antibody titres which were detectable in the ELISA, but no detectable antibody the water-soluble adjuvants or following administration of HTMG applied with the ISU-peptide-KLH conjugate.