Ivermectin in loiasis and concomitant O. volvulus and M. perstans infections.

Two clinical studies were carried out in Gabon, Africa to evaluate the efficacy, safety, and tolerability of ivermectin in the treatment of patients with Loa loa infection. In the first study, 35 patients received single oral doses of ivermectin, 5-200 mcg/kg body weight. Blood microfilariae levels did not decrease after a single oral 5, 10, 30, or 50 mcg/kg dose of ivermectin, but levels did decrease after doses of 100, 150, and 200 mcg/kg. The most efficacious dose was 200 mcg/kg; mean blood microfilariae levels decreased to 12% of mean pretreatment values by day 15 and remained decreased for 28 days. A second study evaluated the safety and efficacy of ivermectin in patients with multifilarial infections. All 17 patients had concomitant Loa loa and O. volvulus infection. M. perstans affected 5 of the patients. Sixteen patients also had infections due to intestinal nematodes. The patients each received single oral doses of 200 mcg/kg ivermectin. Ten days later, the mean Loa loa blood microfilariae level had decreased to 20% of the mean pretreatment level. O. volvulus dermal microfilariae densities were reduced to 2% of the pretreatment levels. A minimal increase in blood microfilaria levels was observed on day 28. In contrast, dermal microfilariae levels remained near zero for the duration of the study. Intestinal infection due to Ascaris was eradicated in all of the affected patients by day 23; efficacy against Trichuris and hookworm infections, however, was poor. All patients tolerated ivermectin well including those with multiple infections.

Expression in Escherichia coli of fully active recombinant human IL 1 beta: comparison with native human IL 1 beta.

Although complementary DNA (cDNA) encoding human interleukin 1 beta (IL 1 beta) have been cloned in several laboratories, there are as yet no data demonstrating that recombinant IL 1 beta (rIL 1 beta) molecules expressed from such cDNA are faithful, fully active replicas of the native protein secreted by human monocytes. To this purpose, cDNA sequences corresponding to the exact NH2-terminus and amino acid sequence of mature, monocyte-derived human IL 1 beta were placed under control of the inducible trp-lac (TAC) fusion promoter and were expressed in E. coli strain JM105. rIL 1 beta was purified to homogeneity by high pressure liquid chromatography (HPLC). Yields of 10 to 20 mg of rIL 1 beta/liter/10(11) cells were obtained. Purified rIL 1 beta was then compared in a number of biochemical and biologic assays to purified native IL 1 beta. Native and rIL 1 beta co-migrated on SDS-polyacrylamide gels as 17.5 kd polypeptides and reacted with specific polyclonal antisera raised to three synthetic peptides of human IL 1 beta in immunoblot experiments. Amino acid sequence analysis of rIL 1 beta showed that the native amino terminus, an ALA residue, was faithfully maintained. Purified native and rIL 1 beta co-chromatographed on reverse-phase HPLC. Specific biologic activities of rIL 1 beta were indistinguishable from those of the native protein in murine thymocyte and human dermal fibroblast proliferation assays, with half-maximal stimulation occurring at concentrations of 25 pM in the murine thymocyte assay and 2 pM in the human dermal fibroblast assay. Similarly, native and rIL 1 beta competed equally well for high affinity IL 1 receptor binding sites, each exhibiting a Ki of 20 pM on MRC-5 human embryonic lung fibroblasts. These observations indicate that E. coli efficiently expresses large quantities of rIL 1 beta, which emulates exactly the properties of the native protein.

Specific bioactivities of monocyte-derived interleukin 1 alpha and interleukin 1 beta are similar to each other on cultured murine thymocytes and on cultured human connective tissue cells.

In this report we compare the bioactivities of pure, human monocyte-derived interleukin 1 (IL-1) alpha and beta in the standard murine thymocyte proliferation assay, a human dermal fibroblast proliferation assay, and in an assay measuring stimulation of prostaglandin E2 (PGE2) release from human rheumatoid synoviocytes. In each case the different species of IL-1 produced saturable stimulation and gave similar dose response curves. Half-maximal stimulation was observed at average IL-1 concentrations of 29 pM in the thymocyte assay, 2 pM in the dermal fibroblast proliferation assay, and 5 pM in the synovial cell assay. Our results show that native, monocyte-derived IL-1 alpha and IL-1 beta are both potent stimulators of connective tissue cells and that the specific bioactivities of these molecules are similar to each other in tests on human connective tissue cells, as well as on murine lymphoid cells.

Immunocytochemical detection of interleukin 1 within stimulated human monocytes.

We have used synthetic peptides coupled to KLH to raise high titer antisera to human IL-1 beta, and in the present report show the usefulness of these sera for immunocytochemical analyses of IL-1 production. Using indirect immunofluorescence, we have been able to specifically identify IL-1 within human monocytes and to monitor its accumulation with time. After indirect immunofluorescent staining of LPS- and PHA-stimulated mononuclear cell cultures, intense cytoplasmic fluorescence was observed in 93% of the monocytes, but not in lymphocytes or platelets present in the same preparation. Unstimulated monocytes did not contain immunocytochemically detectable IL-1. When put into culture, however, some of the otherwise unstimulated monocytes subsequently showed a transient accumulation of intracellular IL-1. Monocytes cultured in the presence of LPS and PHA exhibited detectable fluorescence after 2.5 h, and the fluorescent intensity of these cells continued to increase over the course of 21 h. Fluorescent staining was abolished by preincubation of the sera with relevant but not irrelevant peptide, and while preimmune or anti-KLH serum produced no staining, antisera against either the amino terminus or an internal region of IL-1 beta produced identical staining patterns. Immunoblot analyses of lysates from stimulated monocytes showed that the antisera against IL-1 recognize a single intracellular species with an apparent molecular weight (33 kD) similar to that predicted for IL-1 precursor from the nucleotide sequence of IL-1 cDNA. The ability to specifically identify and immunocytochemically localize IL-1 within producing cells should prove extremely useful for studying the in situ production of IL-1 in immune-based and inflammatory diseases.

Extracellular matrix-cytoskeletal interactions in rheumatoid arthritis. I. Immunoelectron microscopic analysis of the fibronexus at the adhesive surface of normal porcine type B synoviocytes in vitro.

We studied cell surface interactions between the fibronectin (FN)-containing extracellular matrix and the actin cytoskeleton of normal porcine synoviocytes in vitro, using electron microscopic methods. These type B synovial cells were distinguishable from dermal fibroblasts co-isolated from the same organism, because of their very long cellular processes and their ability to synthesize prostaglandin E2 after stimulation with interleukin-1. With plastic sections, we found end-to-end (tandem) and track-like (lateral) transmembrane associations of extracellular fibers and cortical 5-nm microfilaments localized along the attenuated synoviocyte processes in postconfluent cultures. Very similar FN-actin complexes, termed fibronexus (FNX), have been observed on cultured fibroblasts and on granulation tissue myofibroblasts in vivo. Using double-label immunoelectron microscopy with monospecific antibodies applied to ultrathin frozen sections of synoviocytes cut in situ, we proved that these FNX were indeed composed of associated FN and actin filaments. The striking finding of numerous FNX in cultured type B synoviocytes strongly suggests that the FNX is a major cell surface adhesion site in normal synovium, which may play an important role in pannus formation, connective tissue remodeling, and synoviocyte proliferation in patients with rheumatoid arthritis.