Casadevall, A., J. tissue (2). The presence of glucuronoxylomannan (GXM), the major capsular polysaccharide of pathogenesis since this compound has been associated with a variety of immunosuppressive effects (20). For instance, GXM can interfere with phagocytosis, antigen presentation, leukocyte migration and proliferation, and specific antibody responses, and the capsular polysaccharide can enhance HIV replication (20). releases abundant capsular polysaccharide in the supernatant of liquid cultures and in tissues (7, 10). In individuals with impaired immunity affected by cryptococcosis, high levels Chlorogenic acid of capsular polysaccharide are often detected in serum and cerebrospinal Chlorogenic acid fluid. Recent investigations suggest that the accumulation of GXM in the cytoplasmic vacuoles of phagocytic cells might contribute to cell destruction (10). Administration of monoclonal antibody (MAb) directed against GXM significantly reduces serum GXM levels in rodents infected with through the formation of antigen-antibody complexes that are taken up by reticuloendothelial cells (13). MAb to capsular polysaccharide promotes opsonization, phagocytosis, and growth inhibition of encapsulated by phagocytic cells (15). However, we hypothesized that antibody may also reduce GXM levels by interfering with the release of polysaccharide from your capsule. strains H99 (serotype A) and 24067 (serotype D) were acquired from John Perfect (Durham, N.C.) and the American Type Culture Collection (Rockville, Md.), respectively. These strains have been well characterized. MAbs 18B7 (IgG1), 12A1 (IgM), 13F1 (IgM), and 21D2 (IgM) each bind to GXM and have been explained previously (3, 4, 6, 15). The murine IgG1 MAbs 3671 and 3665 and the murine IgM MAb 4F11 were used as isotype-matched controls, having specificity for phenylarsonate and arabinomannan, respectively (9, 19). Neither MAb 3665, 3671, nor 4F11 binds to polysaccharide. MAbs 18B7, 3665, and 3671 were purified by protein G affinity chromatography (Pierce, Rockford, Ill.). IgM MAbs 12A1, 13F1, 21D2, and 4F11 were used as ascites or after mannan-binding protein affinity chromatography (Pierce). Antibody concentration was determined by enzyme-linked immunosorbent assay (ELISA) relative to isotype-matched requirements. capsular GXM was measured by capture ELISA as explained previously (4). Briefly, samples of supernatant to be assayed for GXM were treated with 20 g of proteinase K/ml overnight at 37C to digest MAb before evaluation by capture ELISA. After proteolytic digestion, the samples were heated at 100C for 15 min to inactivate the enzyme. Microtiter polystyrene plates were coated with goat anti-mouse IgM (1 g/ml) and blocked with 1% bovine serum albumin in phosphate-buffered saline. Next, the IgM GXM binding MAb 2D10 (2 g/ml) was added as a capture antibody, and the plate was incubated for 1 h. The solution to be tested for GXM was then added, serially diluted around the plate, and incubated for 1 h. The ELISA was completed by adding, in successive actions, MAb 18B7 (2 g/ml) in phosphate-buffered saline (1% bovine serum albumin), 1 g of alkaline phosphatase-labeled goat anti-mouse IgG1/ml, and 50 l of values of <0.05 were considered significant. Capsule size measurements of cells were carried out for cells produced in the presence and absence of MAb 18B7, as explained previously (21). H99 and 24067 cells were Cldn5 Chlorogenic acid produced in Dulbecco’s altered Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) with MAbs 18B7, 12A1, 13F1, and 21D2 or irrelevant control MAbs 3671, 3665, and 4F11 (100 g/ml) for 48 h at 37C without shaking to prevent antibody-mediated clumping of cells. Capsule size of cells produced in DMEM (10% FCS) culture was measured at 0, 24, and 48 h after incubation. Three microliters of culture of cells produced without MAb and in the presence of MAbs 18B7, 12A1, 13F1, and 21D2 or MAbs 3671, 3665, and 4F11 was placed on a slide, and a small drop of India ink was added. The slides were viewed under a.