No abstract

The Staphylococcus xylosus xyl genes were cloned in Staphylococcus carnosus by complementation to xylose utilization. Xylose isomerase assays under inducing (xylose present) and non-inducing (xylose absent) conditions indicated the presence of a regulated xylA gene on the recombinant plasmid. The nucleotide sequence (4520 bases) revealed three open reading frames with the same polarity. They were identified by sequence homologies as xylR, encoding the Xyl repressor, xylA, encoding xylose isomerase and xylB, encoding xylulokinase. Primer extension analyses indicated constitutive transcription of xylR and xylose-inducible transcription of xylA. Promoter consensus sequences were found upstream of both transcriptional start sites. A transcriptional terminator between xylR and xylA separates the different transcriptional units. Potential regulatory elements were identified by sequence analysis and suggest a repressor-operator mechanism for the regulation of xylAB expression.

Objective. To determine whether systemic elimination of macrophages by means of clodronate‐containing liposomes counteracts inflammation and joint destruction in rats with established adjuvant arthritis (AA). Methods. Rats with AA received a total of 2.7 mg of clodronate encapsulated in liposomes in 3 intravenous doses on days 10, 11, and 12 of arthritis. Phosphate buffered saline (PBS), PBS‐laden liposomes, or free clodronate were used as negative controls. Clinical, hematologic, and histopathologic signs of AA were monitored, and depletion of macrophages by clodronate‐liposomes was evaluated both in the synovial membrane (SM) and in organs of the mononuclear phagocyte system (MPS). Results. Clodronate‐laden liposomes led to significant, long‐term amelioration of the clinical signs of AA, a reduction in the erythrocyte sedimentation rate (ESR), and counteraction of joint destruction, not only immediately after treatment, but also for 2 weeks thereafter. Free clodronate induced moderate clinical improvement and a significant decrease in the ESR, but only during the late phase of AA. Drug‐free vesicles even aggravated the joint destruction. Clodronate‐laden liposomes did not induce significant depletion of resident macrophages in the SM, but rather, in the paracortical region of popliteal lymph nodes, in the liver, and in the marginal zone and periarteriolar lymphatic sheaths of the spleen. Conclusion. Clodronate‐laden liposomes induce long‐term amelioration of AA, even if administered for a brief period during the florid phase of the disease. The amelioration is paralleled by the elimination of macrophages in immunocompetent areas of the spleen and draining lymph nodes, but not locally in the SM. This suggests an influence of the treatment on the immunoregulatory rather than effector, functions of macrophages.

The present study was performed to elucidate whether sterically stabilized liposomes laden with clodronate, which lead to depletion of macrophages (Mphis) and amelioration of experimental autoimmune arthritis in vivo, selectively affect cells of the mphi lineage in vitro. The rates of incorporation of drug-free, fluorescent liposomes and the rates of cell death following exposure to clodronate-liposomes were assessed in human peripheral blood monocytes, as well as in polymorphonuclear leukocytes (PMNs), T cells, endothelial cells, and fibroblasts, both at rest and following activation. Gel electrophoresis of nuclear extracts and ultrastructural analyses were performed to identify the modality of cell death. Monocytes, particularly upon activation, were more efficient in incorporating sterically stabilized liposomes than all other cells except PMNs. Twenty percent of resting monocytes and up to 65% of activated monocytes died within 24 h of exposure to clodronate-liposomes, whereas the other cell types, including PMNs, remained unaffected. Activated monocytes exposed to clodronate-liposomes, but not resting or activated monocytes exposed to drug-free liposomes, showed clear signs of apoptotic cell death. In most of the assays, sterically stabilized liposomes were more efficient than conventional phosphatidylcholine-liposomes. Sterically stabilized clodronate-liposomes preferentially affect cells of the mphi lineage, particularly if activated. Selective elimination of activated Mphis by apoptosis may explain both therapeutic efficacy and safety of clodronate-liposomes in experimental models of autoimmunity.