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This study evaluated the potential pharmacokinetic interaction of pamapimod, a p38 mitogen-activated protein kinase inhibitor, and methotrexate (MTX) when administered concomitantly in patients with rheumatoid arthritis (RA); the study also evaluated the pharmacodynamic effects of pamapimod. Twenty-two RA patients on a stable regimen of MTX (10-25 mg/wk; administered on days 1 and 8) were randomized to receive 300 mg of pamapimod (n = 17) or placebo (n = 5) once daily (qd) for 10 days (days 5-14). Blood and urine samples were collected pre- and postdose on days 1 (MTX alone), 7 (pamapimod alone), and 8 (MTX and pamapimod coadministered). No clinically significant changes were observed in plasma exposures and renal clearance of pamapimod, MTX, or their metabolites, whether administered separately or concomitantly. The combination of pamapimod (300 mg qd) for 10 days and weekly MTX was generally well tolerated. Parameters of RA disease--namely, tender joint count, swollen joint count, erythrocyte sedimentation rate, and C-reactive protein--generally decreased between days 5 and 14. The results of this study suggest that dose adjustments for either drug are not necessary when concomitantly administered and that pamapimod can decrease pharmacodynamic markers of disease activity.

Objective To determine the efficacy and safety of pamapimod (a selective inhibitor of the α-isoform of p38 MAP kinase) as monotherapy in comparison with methotrexate (MTX) treatment in adult patients with active rheumatoid arthritis (RA). Methods Patients were randomly assigned to 1 of 4 treatment groups and received 12 weeks of double-blind treatment. One group received MTX (7.5 mg/week with planned escalation to 20 mg/week), and 3 groups received pamapimod (50, 150, or 300 mg) once daily. The primary efficacy end point was the proportion of patients meeting the American College of Rheumatology 20% improvement criteria (achieving an ACR20 response) at 12 weeks. Secondary end points included ACR50 and ACR70 responses, change from baseline in the Disease Activity Score in 28 joints (DAS28), categorical analyses of DAS28/European League Against Rheumatism response, and change from baseline in each parameter of the ACR core set of measures. Safety monitoring included recording of adverse events (AEs), laboratory testing, immunology assessments, administration of electrocardiograms, and assessment of vital signs. Results Patients assigned to receive MTX and pamapimod had similar demographics and baseline characteristics. At week 12, fewer patients taking pamapimod had an ACR20 response (23%, 18%, and 31% in the 50-, 150-, and 300-mg groups, respectively) compared with patients taking MTX (45%). Secondary efficacy end points showed a similar pattern. AEs were typically characterized as mild and included infections, skin disorders, and dizziness. Pamapimod was generally well tolerated, but the 300-mg dose appeared to be more toxic than either the 2 lower doses or MTX. Conclusion The present results showed that pamapimod was not as effective as MTX in the treatment of active RA.

The granulomatous pathology in human intestinal schistosomiasis is induced primarily by the egg antigens of schistosome, a parasitic trematode. Glycolipids and glycoproteins were extracted from the eggs of the two major species which infect human, Schistosoma mansoni and Schistosoma japonicum, for structural characterization based on highly sensitive mass spectrometric analysis coupled with chemical derivatization. Here, we demonstrate that a series of uniquely multifucosylated glycosphingolipids constitute the major egg glycolipids of S. mansoni but not of S. japonicum. The S. mansoni glycosphingolipids were found to be extended by varying numbers of an unusual repeating unit, -->4(Fuc1-->2Fuc1-->3)GlcNAc1-->, and terminating as +/-Fuc1-->2Fuc1-->3GalNAc1--> at the nonreducing terminus. The similarity of these fucosylated structures, particularly the nonreducing terminal sequence, to the previously identified multifucosylated O-linked oligosaccharides of the cercarial glycocalyx, suggests that they may constitute the cross-reacting epitopes between the egg antigens and cercariae of S. mansoni. On the other hand, the difucosylated GalNAc terminal epitope was not found on the glycosphingolipids of S. japonicum. Thus, it qualifies for a possible role as a species-specific recognition glycan.

The entire surface of the cercarial stage of the human blood fluke Schistosoma mansoni is covered by a 1-microns thick, highly immunogenic, fucose-rich glycocalyx (GCX). Using strategies based on enzymatic, chemical, and mass spectrometric analysis, we have defined the structures of the major glycans released by reductive elimination from GCX. They comprise a heterogeneous population of multifocosylated complex oligosaccharides with the following nonreducing terminal sequences: [formula: see text] Our structural data suggest that these tri- to pentafucosylated epitopes are carried on type 1, R--Gal beta-1--3GalNAc, and type 2, R--Gal beta 1--3(R--GlcNAc beta-1--6)GalNAc, core structures via repeat units of (3GalNAc beta 1--4(Fuc alpha 1--2Fuc alpha 1--2Fuc alpha 1--3)GlcNAc beta-1--3Gal alpha--)n, where n is mainly 0 and 1, and all sugars are in the pyranose form. The proposed structure represents the first instance where an alpha-galactosylated beta-GalNAc(1--4)-beta-GlcNAc sequence occurs as a repeating unit in a glycoprotein. It is also unique in being substituted with oligofucosyl appendages. The unusual oligosaccharide structures described here, particularly the potentially immunodominant oligofucosyl moieties, are most likely responsible for the known potency of GCX in modulating various immune responses including complement activation, B cell mitogenesis, and delayed type hypersensitivity in schistosomiasis.

To determine the consequences of mast cell (MC)-chondrocyte interactions.Cocultured cells were analyzed histochemically, morphologically, biochemically, and functionally.Cocultured MC adhered to the chondrocytes and remained viable. Chondrocytes cocultured with nonactivated MC produced more proteoglycans than did chondrocytes cultured alone, and these proteoglycans possessed an intact hyaluronic acid-binding region. In contrast, most of the proteoglycans produced by chondrocytes cocultured with activated MC were degraded.These studies indicate that a complex interaction occurs in which the nonactivated MC stimulates biosynthesis and the activated MC degrades cartilage proteoglycans.

Objective:To determine the efficacy and safety of pamapimod in adult patients with active rheumatoid arthritis (RA) who had an inadequate clinical response to methotrexate (MTX).Methods:Patients receiving stable doses of MTX were randomised to one of six dose groups and received 12 weeks of double-blind pamapimod (up to 300 mg once daily) or matching placebo. The primary efficacy measure was the proportion of patients with ⩾20% improvement in RA based on the American College of Rheumatology criteria (ACR20) at 12 weeks. Secondary measures were ACR50, Disease Activity Score (DAS)/European League Against Rheumatism (EULAR) responses and the individual ACR core set of parameters. Safety measures included adverse events (AEs), laboratory testing and immunology assessments.Results:On a background of MTX, the percentage of patients with an ACR20 response at week 12 in the pamapimod groups (31% to 43%) was not significantly different from placebo (34%). Secondary efficacy end points showed a similar pattern. AEs were typically mild and included infections, gastrointestinal disturbances, dizziness and rashes; AEs resulting in discontinuation of study drug were primarily attributed to infections.Conclusion:In patients with active RA receiving stable doses of MTX, pamapimod showed non-significant improvement in efficacy outcomes compared to placebo.

This study compares 4 baseline correction methods on the effect of moxifloxacin on the QT/QTc interval: (1) day -1 time-matched baseline electrocardiograms (ECGs), (2) 3 triplicate predose ECGs, (3) 1 triplicate predose ECG, and (4) no baseline correction. Forty-four healthy subjects receive a single dose of moxifloxacin (400 mg), placebo, and 2 doses of an investigational agent in a 4-period crossover fashion. For all 4 methods, the largest mean difference from placebo in the moxifloxacin study-specific QTc is 11.97 to 13.23 ms and occurs at 3 to 4 hours postdose; the lower 90% confidence interval is greater than 5 ms from 2 to 8 hours. The average standard error of the mean is 1.36 ms for 3 triplicate predose ECGs, 1.40 ms for 1 triplicate predose ECG, 1.60 ms for day -1 time-matched baseline ECGs, and 1.65 ms for no baseline correction. Predose baseline methods (3 or 1 triplicate ECGs) are superior to the day -1 time-matched baseline correction or without baseline correction.

Large granular lymphocytes, mediators of NK activity, bind to other cells using both the LFA (lymphocyte function-associated)-1-ICAM and the CD2-LFA-3 adhesion pathways. Here we have studied the motility and ultrastructure of large granule lymphocyte (LGL) on lipid bilayers containing purified LFA-1, ICAM-1, and the transmembrane and glycophosphatidylinositol isoforms of LFA-3. LGLs moved at 8 microns/min on ICAM-1 but poorly (less than 1 microns/min) on its receptor pair LFA-1. TM-LFA-3 promoted locomotion at a rate close to ICAM-1, whereas the cells were less motile on GPI-LFA-3. The difference in the rates of locomotion on the two isoforms of LFA-3 is presumably attributable to their difference in anchoring and lateral mobility in the bilayer. In spite of the variation in motility the ultrastructure of the adhering cells was similar on all four ligands. LGLs contacted the membrane variably, i.e., cells adhering only in a few small areas or in larger areas were detected on each ligand. The relative percentage of the plasma membrane facing the lipid bilayer was greatest on ICAM-1 and least on the transmembrane isoform of LFA-3, demonstrating no correlation with motility. The ratio of adjacent plasma membrane to lipid bilayer was virtually constant for all four ligands. Activation of the LGLs with a combination of CD2 mAb T11(2) and T11(3) (T11(2/3) mAb) reduced the movement on ICAM-1 and virtually immobilized the cells on the other bilayers. In the presence of T11(2/3) mAb, the area of cell membrane attaching to bilayers containing ICAM-1 and GPI-LFA-3 was decreased and the percentage of plasma membrane facing other cells was increased. No preferential orientation of the Golgi apparatus or degranulation was detected in the absence or presence of T11(2/3) mAb, but a significantly lower percentage of LGLs on ICAM-1 contained a profile of the Golgi apparatus after exposure to T11(2/3) mAb. The results demonstrate that the motility of LGLs depends on the type of receptor in the opposing bilayer, the receptor mobility in the bilayer, and the activation of the cells. The ultrastructure of LGLs binding to any of the adhesion molecules does not have the characteristics of LGLs in cytolytic contact with target cells, suggesting that the mediation of an attack on a target requires more complex stimulus than any one of the single adhesion proteins tested here.

Unlike human mast cells associated with mucosal surfaces such as lung, adenoids, tonsils and intestine, skin mast cells may be stimulated to release histamine by the neuropeptides substance P, vaso-active intestinal polypeptide and somatostatin or by other basic secretagogues such as morphine and compound 48/80. Release of histamine by neuropeptides is rapid and accompanied by minimal generation of the eicosanoids prostaglandin D2 and leukotriene C4. Transient elevations of intracellular calcium are associated with mediator secretion induced by both immunological and non-immunological stimulation, that induced by anti-IgE being derived from extracellular sources through channels in the plasma membrane while that stimulated by neuropeptides is mobilized intracellulary. Similarly, elevations of intracellular cyclic AMP induced by anti-IgE occur only in the presence of extracellular calcium, whereas with substance P elevations are apparent even in the absence of extracellular calcium. With the latter stimulus, histamine release is complete before the peak cyclic AMP is achieved. Despite these biochemical and temporal differences, degranulation induced by both secretagogues proceeds by compound exocytosis which is indistinguishable under the electron microscope. From these results we suggest that IgE-dependent and neuropeptide stimulation of human skin mast cells proceed by distinct biochemical pathways which eventually merge to produce exocytosis of their preformed granule-associated mediators.

Background. Mycophenolic acid (MPA), a selective inhibitor of inosine monophosphate dehydrogenase, is the active agent of the immunosuppressive drug, mycophenolate mofetil (MMF). Previous studies have shown that MPA inhibits DNA synthesis in T and B lymphocytes by blocking de novo guanosine synthesis, and that MPA induces monocyte differentiation. MMF is being used for prevention of organ graft rejection and has also shown efficacy in rheumatoid arthritis trials. This study was designed to determine if apoptosis also plays a role in the immunosuppressive and anti-inflammatory effects of MMF. Methods. Cultured human T lymphocytic (MOLT-4) and monocytic (THP-1 and U937) cell lines were treated with MPA. Apoptosis, cell viability, DNA content, lipid content, cell volume, and lysosomes were measured by a variety of microscopic, flow cytometric, and biochemical techniques. Results. MPA inhibits proliferation, arrests cell cycle in S phase, and increases apoptosis in all three cell lines. Exogenous guanosine added within 24 hr of MPA treatment, but not later, partially reversed MPA-induced apoptosis in MOLT-4 cells. MPA increased lipid droplets in all three cell lines and increased both cell volumes and numbers of lysosomes in the monocytic cell lines. In both monocytic cell lines, MPA also reduced the number of nuclei containing nucleoli and greatly increased neutral lipids, primarily triacylglycerols, suggesting that these cells were differentiating. Conclusions. Increased apoptosis and terminal differentiation of both lymphocytes and monocytes may promote the antiproliferative, immunosuppressive, and anti-inflammatory effects of MMF seen clinically in transplantation and rheumatoid arthritis.

The structural diversity of the glycans from Schistosoma mansoni and Schistosoma japonicum egg glycoproteins was investigated using high sensitivity fast atom bombardment mass spectrometric screening of glycan pools released enzymically or chemically from egg extracts. The egg glycoproteins from the two species carry a comparable range of high mannose and complex type N-glycans with both lacNAc and lacdiNAc constituting the backbones of the antennae in the latter class. Truncated N-glycans similar to those found on nematodes, insects, and plants were also identified. Sequential digestion with peptide N-glycosidase F and peptide N-glycosidase A afforded effective release and separation of N-glycans with nonfucosylated or alpha6-monofucosylated trimannosyl N,N'-diacetyl-chitobiose cores from those carrying core alpha3-, alpha6-difucosylation, all of which were found to be present in both species. Remarkably, a portion of the N-glycans from S. mansoni eggs was shown to be based on a xylosylated, alpha6-fucosylated trimannosyl core, whereas a portion of those from S. japonicum contains a xylosylated alpha3-, alpha6-difucosylated core which has not been previously described in any organism. O-Glycans were chemically released from the de-N-glycosylated glycopeptides and found to carry terminal sequences similar to those in the N-glycans. This study provides further evidence that multi-fucosylated terminal HexNAc units, previously identified on the cercarial glycocalyx O-glycans and egg glycosphingolipids, and now on the egg N- and O-glycans, are unique features of S. mansoni glycans. These multifucosylated terminal structures, which were not detected on the egg glycans of S. japonicum, are likely to constitute the cross-reacting epitopes between the eggs and cercariae of S. mansoni. Interestingly other HexNAc termini, including an unusual stretch of HexNAc3, were found to be common to both species. The mapping studies reported in this article provide an important foundation for further structural work in this challenging and important area of glycobiology.

It was predicted from the amino acid sequence of the bone anabolic peptides, parathyroid hormone (PTH) (1-34) and PTH related protein (PTHrP) (1-34), that the C-terminal amino acids form an amphipathic alpha-helix. Therefore, we substituted a model amphipathic alpha-helical peptide (MAP) sequence in the C-terminal region of hPTHrP(1-34), obtaining RS-66271 ([MAP1-10]22-31 hPTHrP(1-34)-NH2). The anabolic activities of RS-66271 and hPTHrP(1-34) were evaluated in 3-month-old, ovariectomized (OVX) osteopenic rats. Subcutaneous injection of hPTHrP(1-34) at 80 micrograms/kg/day partially reversed estrogen depletion trabecular bone loss but was ineffective in the cortex. In contrast, RS-66271 dose-relatedly reversed loss at both sites and, at 80 micrograms/kg/day, returned both trabecular and cortical bone calcium to the level of sham-operated controls. Histomorphometric analysis showed significantly elevated bone formation rates over vehicle-treated OVX in both trabecular and cortical tibial bone following treatment with RS-66271. Electron microscopy showed an increase in the relative surface area of vertebral trabeculae covered by osteoblasts in animals treated with RS-66271. These studies demonstrate that the C-terminal amino acids of hPTHrP(1-34) can be replaced by a model amphipathic helix and that the new chemical entity has greater anabolic activity than the parent peptide. The results suggest that RS-66271 may be a candidate molecule for the treatment of human osteoporosis.

Schistosomes metabolize large quantities of glucose obtained from the host serum by facilitated diffusion through the tegument. Here we have used rabbit antibodies affinity purified against the carboxyl terminus of a facilitated glucose transporter, SGTP4, to localize the antigen in both schistosomula and adults. By ultrastructural immunocytochemical analysis, SGTP4 was localized to both lipid bilayers that cover the tegumental surface of adults and schistosomula. In the inner bilayer of adults, SGTP4 was apparently oriented with the carboxyl terminus on the internal side of the bilayer. SGTP4 was also present in the discoid and multilamellar bodies in adults and the membranous bodies in schistosomula. Finally, the affinity purified antibodies against SGTP4 bound nonspecifically to the head glands and postacetabular glands in schistosomula. The localization of the antigen in the two surface lipid bilayers suggests that SGTP4 may be responsible for transporting glucose from mammalian host serum into the tegument.

RS-23581, a synthetic analog of human PTH-related protein-(1-34), and the amino-terminal 34 amino acids of bovine PTH [bPTH-(1-34)] increase bone mineral density. We wished to determine how quickly the ultrastructure of the osteogenic cells, i.e. osteoblasts and lining cells, of the cancellous bone of the second lumbar vertebra of ovariectomized rats was altered in response to the initiation and cessation of treatment. Ovariectomized rats were injected daily with 80 micrograms/kg RS-23581, bPTH-(1-34), or vehicle for 19 days. Animals were killed throughout the treatment period and during the ensuing 10 days. By 5 days after the initiation of treatment with either peptide, the cells on the trabecular surface were predominantly (90%) osteoblasts, with only a small increase in the total cell number. Throughout the dosing period, the relative area of the cytoplasm of osteogenic cells from rats treated with RS-23581 or bPTH-(1-34) was greater than that of cells from the ovariectomized control group, suggesting a relationship between bone formation and cytoplasmic mass. By 7 days after the cessation of treatment, the trabecular surface was covered predominantly by lining cells without a change in cell number. Thus, these peptides apparently promote the osteoblast phenotype; the osteoblasts revert to lining cells after the peptides are withdrawn.

SUMMARYAdult parasites ofSchistosoma mansonireside within vertebrate mesenteric veins where they consume immense quantities of host glucose after transporting the sugar through their surface syncytium or tegument. Previously we obtained cDNA clones encoding two functional facilitated diffusion glucose transporter proteins expressed byS. mansoniadult worms (Skellyet al.1994). Antibodies specific for one transporter (SGTP1) have been generated against an extrafacial and an internal domain of the protein and used to localize the protein by light and electron microscopy. By light microscopy both antibodies stain a linear structure approximately 1–5 μm from the surface of the tegument of adult male and female schistosomes. Electron microscopic examination of frozen thin sections show binding of the antibodies to membranes in the base of the tegument and not to the membranes covering the outer surface or their invaginations. Analysis of the gold distribution suggests that the extrafacial domain is disposed toward the interstitial space beneath the tegument and the internal domain faces the syncytial plasm. The localization suggests that SGTP1 may function to transport free glucose from within the tegument and into the interstitial fluids that bathe the internal organs of these parasites.

Schistosomula of Schistosoma mansoni develop the ability to ingest and digest red blood cells after the fourth day post-transformation. Here, we have used fluorescently-labeled dextrans and two plasma proteins, albumin and IgG, to test whether day-old schistosomula can ingest and process macromolecules prior to the time that they eat red cells. Worms ingested dextrans of molecular weights 4,000, 70,000 and 2 x 10(6) in a time- and concentration-dependent manner. The dextran remained in the cecal lumen for up to 2 days after feeding. Parasites ingested both fluorescein-conjugated bovine serum albumin and rabbit IgG, but neither of these proteins remained confined to the cecum over time. Instead, fluorescence redistributed to the acetabular glands within a few hours. Thin-layer chromatography indicated that schistosomula degraded fluorescein-conjugated albumin to fluorescein-conjugated peptides approximately 10-15 amino acids long. The volume of the cecum was estimated to be 2431 microns 3 and the surface area 299 microns 2. These results demonstrate that larval schistosomes can ingest both proteins and complex carbohydrates shortly after transformation, before they can ingest red cells. Further, the gut apparently releases proteases that cleave plasma proteins, but not saccharidases that cleave dextran.

Summary Schistosomula of Schistosoma mansoni bind human low density lipoproteins (LDL) in a concentrationdependent and saturable manner. The bound LDL could provide phospholipids and sterol to the worm, which cannot synthesize sterol de novo and lacks acyl chain-modifying capability. Here we have used three phospholipid analogues to explore the effect of LDL binding on the parasite's outer tegumental membrane, i.e. the outer of the two membranes that cover its surface syncytium. Fluorescein phosphatidylethanolamine (Fl-PE) and rhodamine phosphatddylethanolamine (Rh-PE) bound to the parasite in a saturable manner and, as shown by fluorescence microscopy, were confined to the surface. Fl-PE fluorescence was completely quenched by Trypan Blue and Fl-PE was lost from the surface following single exponential decay kinetics (tj=12h), further suggesting that the probe was confined to the outer membrane. 1,1' -Dioctadecy 1-3,3,3' ,3' -tetramethylindocarbocyanine perchlorate (Dil-C18(3); Dil) did not bind saturably and was seen in both the surface and the internal parasite membranes. Fluorescence photobleaching recovery was used to measure the lateral mobility of Fl-PE in the outer membrane. The lateral diffusion coefficient of Fl-PE was approximately 10" 7 cm 2 s~ 1 . The fractional mobility of Fl-PE was 85 % when measured using a laser beam of radius 1.8 fan and 45% using a beam of radius 4.3/an. These measurements suggest that the outer membrane is composed of /an-scale liquid crystalline-phase lipid domains that lack significant amounts of transmembrane proteins. LDL binding to the parasite surface did not alter the lateral mobility of Fl-PE or the rate of loss of either Fl-PE or Rh-PE. These studies suggest that the binding of LDL to the outer tegumental membrane of schistosomula does not change either the organization of the membrane lipids or their rate of loss from the membrane. If LDL do provide lipids to schistosomula, then direct insertion of lipid molecules into the outer tegumental membrane is an unlikely mechanism for lipid transfer.

Human neutrophils and eosinophils adhere to the surface of schistosomula of Schistosoma mansoni that have been preincubated with antischistosomular sera with or without complement. Neutrophils are seen to form small (< 0.5 micrometer), heptalaminar and large (5-8 micrometer), pentalaminar fusions with the normal pentalaminar parasite surface membrane. By freeze-fracture techniques, attachment areas 5-8 micrometer in diameter are seen to form between neutrophils and schistosomula. These areas have three zones--an edge and two centrally located areas, one of which is rich and one of which is poor in intramembrane particles (IMPs). The edge zone is continuous around the attachment areas and is usually composed of a skip-fracture that passes out of the schistosomular outer membrane into the inner membrane. In some cases, the edge zone is made up of a string of IMPs. The IMP-rich central areas have an IMP concentration similar to that of unattached neutrophil membranes, are raised off of the surface of the schistosomulum, and have two normal schistosomular membranes underneath indicating that they are indeed unattached. the IMP-poor central areas are composed of a fused or hybrid membrane that is continuous with the neutrophil plasma membrane but that bears the same spatial relationship to the schistosomular inner membrane that the normal outer membrane does. Similar changes are seen in samples prepared with glycerination. Eosinophils generally do not fuse with the schistosomular outer membrane but, instead, discharge their granular contents onto the surface of the schistosomula and appear to adhere to the parasite through this discharged material. It is suggested that schistosomula have a capability to fuse with mammalian cells and that this fusion proceeds from a fusion of the outer leaflets to a fusion of the bilayers, as appears also to be the case in other systems.

Lipids in the two surface membranes of Schistosoma mansoni may play an important role in the parasite's defense against host immunity. In particular, lysophosphatidylcholine lyses erythrocytes attached to the parasite and alters the lateral mobilities of their membrane proteins and lipids (Golan et al. 1986). Here, we have studied the incorporation of radiolabeled precursors into the major lipid classes of schistosomula as well as into lipids released by schistosomula into the medium. Radiolabeled polar head groups (choline and ethanolamine) and fatty acid precursors (palmitate and oleate) were linearly incorporated into parasite phospholipids. Fatty acids were differentially incorporated into the various phospholipid classes, principally into phosphatidylcholine and, to a lesser extent, into phosphatidylethanolamine, lysophosphatidylcholine, and phosphatidylserine. The major neutral lipid class labeled, triglycerides, had a decrease in specific activity with time after pulse labeling and the specific activity of the phospholipids increased with time. Thus, triglycerides may provide acyl chains for phospholipid synthesis. Choline was incorporated into phosphatidylcholine and lysophosphatidylcholine, and ethanolamine into phosphatidylethanolamine and lysophosphatidylethanolamine. No evidence was found for phospholipid methylation or demethylation in schistosomula. Labeled lipids were linearly and selectively released into the medium. Triglycerides were released at the highest rate with measurable quantities of phosphatidylcholine, lysophosphatidylcholine, and phosphatidylethanolamine also observed. Monopalmitoylphosphatidylcholine was the only lysophosphatidylcholine present in the medium as demonstrated by reversed-phase chromatography of released choline-labeled lysophosphatidylcholine. These studies demonstrate that schistosomula synthesize phospholipids and neutral lipids and release some of them into the culture medium. In particular, they release a single molecular species of a potent biologically active molecule, monopalmitoylphosphatidylcholine, that may play a role in the parasite's evasion of the immune response.

The development of schistosomula during the first 4 days after transformation from cercariae has been examined in parasites isolated from the lungs of mice and in organisms cultured in lactalbumin and rabbit serum or in the defined serum-free medium, RPMI 1640. The development of organisms grown under all three conditions was the same. Schistosomula increased in length from 67 to 110 μm and decreased in width from 24 to 18 μm, so that the volume remained constant at approximately 2.7 × 104 μm3. The increase in length occurred mainly in the torso or posterior three-quarters of the worm which increased from 49 to 88 μm or 80%, whereas the head increased from 18 to 22 μm or 22%. The spines were lost from the surface that was most rapidly lengthening by gradual resorption into the tegument and were replaced by pits mainly during the first 3 days. These changes resulted in a 325% increase in the surface area of the schistosomula, from 1.2 × 104 to 3.9 × 104 μm2. In addition, the openings of the acetabular ducts, the ventral sucker, and the tail socket all became smaller and flatter over the four-day period. Internally, the major changes were the loss of the acetabular ducts in the pre- and post-acetabular glands and an increase in size of the caecum. In summary, these experiments show that the surface of the schistosomulum is extensively remodeled before intravascular migration occurs and demonstrate the efficacy of RPMI 1640 as a culture medium for schistosomula in the first 4 days after transformation.

Neutrophils and eosinophils adhering to the surface of schistosomula of Schistosoma mansoni have been partially or completely detached with hypertonic sucrose or by pipetting. The sucrose-treated neutrophils are attached only in areas where there are pentalaminar fusions between the neutrophil and tegumental membranes, suggesting that these fusions attach the cells to the parasites. Pipetting breaks many of the attached cells. In thin section, the tegumental membrane underlying these cells is seen to be pentalaminar. By freeze-fracture techniques, modified attachment areas are found. The edge zone often appears as a single strand of intramembrane particles (IMPs) on the P2 face and as a groove on the E2 face. The edge zone may also have large discontinuities, in which case it no longer separates membrane faces of unequal IMP density from one another. In addition, the IMPs on the IMP-rich areas become aggregated and surrounded by craters in the membrane. These experiments suggest that the fusions may be the mechanism by which the parasite acquires some host membrane components on its surface. On the other hand, eosinophil plasma membranes are seen adhering to a layer of electron-dense material on the parasite after the cells have been disrupted by pipetting. This suggests that eosinophils adhere to the parasite surface through their discharged granule material and not by membrane fusions.

The giardins are a group of 29-38-kD proteins in the ventral disk of the protozoan parasite Giardia lamblia. The disk attaches the parasite to the host's intestinal epithelium and is composed of parallel, coiled microtubules that are adjacent to the ventral plasma membrane and from which processes called microribbons extend into the cytoplasm; the microribbons are connected by crossbridges. G. lamblia cytoskeletons, consisting of disks and attached flagella, were isolated and used to show that the 29-38-kD proteins separate into five bands by one-dimensional electrophoresis and into 23 species by two-dimensional analysis. Rabbit antibodies raised against a 33-kD protein band, purified by one-dimensional gel electrophoresis and shown to contain three proteins by two-dimensional electrophoresis, recognized 17 proteins by two-dimensional immunoblot analysis. By immunofluorescence these antibodies reacted with the ventral disk but not with the flagella in isolated cytoskeletons. Electron microscopy revealed that the anti-giardin antibodies bound to the edges of the microribbons but not to the microtubules, crossbridges, or other, nondisk structures. Antibodies to tubulin reacted with both the disk and flagella in isolated cytoskeletons but bound only to the microtubules in these structures. The amino-terminal sequence of the 33-kD immunogen was determined and used to construct a DNA oligomer, and the oligomer was used to isolate the alpha giardin gene. The gene was used to hybrid select RNA, and the in vitro translation product from this RNA was precipitated by the antibodies against the 33-kD immunogen. The gene sequence was a single open reading frame of 885 nucleotides that predicted a protein of 33.8 kD. The protein sequence is unique, having no significant homology to two other giardin sequences or to any sequences within the Protein Identification Resource. It is predicted to be 82% alpha helical. The downstream sequence of the gene indicates that the sequence AGT-PuAA is located six to nine nucleotides beyond the stop codon in all protein-encoding genes of G. lamblia that have been sequenced and reported to date.

The lectin concanavalin A (Con A) was used as a model probe to study the behavior of molecules bound to the surface of recently transformed schistosomula of Schistosoma mansoni. Con A binding was saturable (150-180 pg/organism) and specifically competed by alpha-methyl mannoside. Both FITC-Con A and 125-I-Con A were lost from the surface of schistosomula with a halftime of 8-10 h in culture in defined medium. A comparable decrease in the binding of Con A to schistosomula cultured and then labeled with the lectin indicated that the labeling procedure itself was not inducing the observed change. Internalization of Con A was not seen by either fluorescence microscopy or electron microscope radioautography. In addition, 70-80% of the radioactivity lost from the parasite was recoverable by TCA precipitation from the culture medium as intact Con A (27,000 mol wt on SDS PAGE). Thus, the mechanism of clearance of bound Con A from the surface of cultured schistosomula is apparently by sloughing of Con A molecules intact into the culture media and not by endocytosis and degradation. Con A binding sites, visualized with hemocyanin by scanning electron microscopy, appeared homogeneously distributed over the surface of schistosomula when organisms were labeled at 4 degree C or after fixation with glutaraldehyde. However, Con A and hemocyanin formed aggregates on the surface of schistosomula when labeling was performed at 37 degrees C, which suggests that lectin binding sites have lateral mobility within the plane of the membrane. These aggregates are likely independent of metabolism by the parasite because aggregation also occurs on the surface of organisms killed with azide.

Human red blood cells (RBCs) adhere to and are lysed by schistosomula of Schistosoma mansoni. We have investigated the mechanism of RBC lysis by comparing the dynamic properties of transmembrane protein and lipid probes in adherent ghost membranes with those in control RBCs and in RBCs treated with various membrane perturbants. Fluorescence photobleaching recovery was used to measure the lateral mobility of two integral membrane proteins, glycophorin and band 3, and two lipid analogues, fluorescein phosphatidylethanolamine (Fl-PE) and carbocyanine dyes, in RBCs and ghosts adherent to schistosomula. Adherent ghosts manifested 95-100% immobilization of both membrane proteins and 45-55% immobilization of both lipid probes. In separate experiments, diamide-induced cross-linking of RBC cytoskeletal proteins slowed transmembrane protein diffusion by 30-40%, without affecting either transmembrane protein fractional mobility or lipid probe lateral mobility. Wheat germ agglutinin- and polylysine-induced cross-linking of glycophorin at the extracellular surface caused 80-95% immobilization of the transmembrane proteins, without affecting the fractional mobility of the lipid probe. Egg lysophosphatidylcholine (lysoPC) induced both lysis of RBCs and a concentration-dependent decrease in the lateral mobility of glycophorin, band 3, and Fl-PE in ghost membranes. At a concentration of 8.4 micrograms/ml, lysoPC caused a pattern of protein and lipid immobilization in RBC ghosts identical to that in ghosts adherent to schistosomula. Schistosomula incubated with labeled palmitate released lysoPC into the culture medium at a rate of 1.5 fmol/h per 10(3) organisms. These data suggest that lysoPC is transferred from schistosomula to adherent RBCs, causing their lysis.

Iron-, manganese-, or magnesium-deficiency has been induced in Euglena gracilis. Each arrests cell proliferation, decreases the intracellular content of the deficient metal, and increases that of several other metals. Light and electron microscopy of stationary phase cells reveal that Fe-deficient (-Fe) cells are similar in size and shape to control organisms. Magnesium-deficient (-Mg) cells, however, are larger, and approximately 14% are multilobed, containing 2 to 12 lobes of equal size emanating from a central region. Individual (-Mg) cells and each lobe of multilobed cells contain a single nucleus. Manganese-deficient (-Mn) organisms are morphologically more heterogeneous than (-Fe) or (-Mg) cells. Most are spherical and larger than controls. Approximately 15% are multilobed but, unlike (-Mg) cells, contain lobes of unequal size with either zero, one, or several nuclei present in each. Nuclei of (-Mn) cells differ in size and shape from those of control, (-Fe), or (-Mg) cells. All three deficient cell types accumulate large quantities of paramylon. Other cytoplasmic structures, however, appear normal. Addition of Fe, Mn, or Mg to the respective deficient stationary phase cultures reverses growth arrest and restores normal morphology. The results suggest that Fe-, Mn-, and Mg-deficiencies affect different stages of the E. gracilis cell cycle.

Lysozyme (pI = II) has been used to identify anionic sites in the glomerular capillary wall. A solution of 1-3% lysozyme was perfused into the left kidney at varying rates. After perfusion, the kidney was fixed in situ and processed for electron microscopy. Lysozyme was seen as an electron-dense deposit which was not present when succinylated lysozyme (pI = 4.5) or myoglobin (pI = 6.9) was perfused instead of native lysozyme. First, the epithelial cell plasma membrane was outlined by a 300-400 A electron-dense layer. Second, there were discrete dense deposits in the subepithelial portions (lamina rara externa) of the basement membrane which, in normal sections, extended from the epithelial cell membrane to the lamina densa and, in grazing section, formed a continuous reticular pattern.

Flagella-specific proteins of Leishmania have been identified employing the monoclonal antibody technique. Six monoclonal antibodies recognized 3 different proteins. A doublet of protein of Mr 69,000 and 74,000 Da identified by monoclonal antibodies F-3, F-4 and F-6 is continuously distributed along the flagellum by immunofluorescence. Immunocytochemical electron microscopic studies localize these molecules to the paraxial rod of the flagellum. A single protein of Mr 13,200 Da is recognized by monoclonal antibodies F-1, F-2 and F-5. The distribution of the Mr 13,200 protein appears irregular, occurring in localized patches along the length of the flagellum, especially at the flagellar tip. Immunocytochemical electron microscopic experiments show that the Mr 13,200 molecule is associated with the membrane of the flagellum. Indirect immunofluorescence experiments demonstrated these monoclonal antibodies cross-reacted with members of the Kinetoplastida family (Endotrypanum, trypanosoma, Leishmania) suggesting that these molecules may be evolutionarily conserved.

We studied the adherence of human erythrocytes to larvae of the intravascular parasite Schistosoma mansoni by transmission microscopy, freeze fracture, and fluorescence techniques. In addition, we used the adherent cells to investigate the problem of host antigen acquisition. Schistosomula were cultured for from 24 to 48 h after transformation in order to clear the remnants of the cercarial glycocalyx. In some cases, the worms were preincubated with wheat germ agglutinin to promote adherence of the erythrocytes. The results were similar with and without the lectin except that more cells attached to the lectin-coated parasites. Erythrocytes adhered within a few hours and, unlike neutrophils, did not fuse with the parasite. A layer of 10-20-nm electron dense material separated the outer leaflets of the tegumental and plasma membranes. In addition, many deformed and lysed cells were seen on the parasite surface. The ability of the worm to acquire erythrocyte membrane constituents was tested with carbocyanine dyes, fluorescein covalently conjugated to glycophorin, monoclonal antibodies against B and H blood group glycolipids, and rabbit alpha-human erythrocyte IgG. In summary, glycophorin, erythrocyte proteins, and glycolipids were not transferred to the parasite membrane within 48 h. Carbocyanine dyes were rapidly transferred to the parasite with or without lectin preincubation. Thus, the dye in the worm membrane came from both adherent and nonadherent cells. These studies suggest that, in the absence of membrane fusion, the parasite may acquire some lipid molecules similar in structure to host membrane glycolipids by simple transfer through the medium but that B and H glycolipids and erythrocyte membrane proteins are not transferred from adhering cells to the worm.

The development of the cercarial glycocalyx of Schistosoma mansoni was studied by transmission electron microscopy and immunofluorescence light microscopy employing antibodies raised against extracted and chromatographed glycocalyx. By electron microscopy, cercariae present in the brood chamber of daughter sporocysts were surrounded by an electron-dense granular and fibrillar matrix. This material appeared structurally distinct from the glycocalyx which was coarsely fibrillar and located only on the surface of organisms that had developed a final tegument. The thickness of the glycocalyx apparently increased with the maturation of the tegument, since teguments that had many spines also had the thickest glycocalyx. Immunofluorescent staining of frozen sections of infected snail hepatopancreas showed that glycocalyx antigens were present on the surface of the cercariae and not in the matrix within the brood chamber or in snail tissues. Immunofluorescent staining of isolated larval cercariae showed staining of some but not all parasites with partially elongated tails. These studies suggest that the glycocalyx develops late in cercarial development (late in Stage 6 or in Stage 7 of Cheng and Bier), is made by the cercariae themselves, and is not a product of either the sporocyst wall cells or snail hepatopancreas.

Purified rat peritoneal mast cells contained 3.3 × 10−5 gm SO4 and 2.2 × 10−8 gm Ca/106 cells. The molar ratio of S/Ca in the whole cell was 600:1. Frozen thin sections of unfixed mast cells contained only sulfur (S) in the granules when examined by X-ray energy dispersive spectroscopy (EDS). Mast cells fixed in 3% glutaraldehyde and 1.5% formaldehyde in 75% ethanol (Et/Ald) or in mixed buffered aldehydes and embedded in Epon 812 or the low viscosity resin diepoxyoctane (DEO) contained S in all granules and Ca in some of the granules measured. Neither element was found in the nucleus, cytoplasm, or resin. Isolated, Et/Ald fixed and embedded granules also contained S. The presence of Ca in the granules was artifactual in that the Ca was absorbed from water in the trough of the diamond knife and/or from the filter paper used to blot the sections dry. This phenomenon was investigated further. Sections of Et/Ald fixed and embedded mast cells were incubated with 5 × 10−6 to 10−2 M CaCl2. Ca was detectable in 100% of the granules incubated at concentrations ≥ 10−4 M and reached a constant S/Ca ratio of 2.0 at concentrations ≥ 10−3 M. Ca was not detectable in the nucleus, cytoplasm, or resin at 10−2 M. A plot of S versus Ca counts from the granules of cells incubated with 10−2 M CaCl2 was linear with a slope of 2.0 and a correlation coefficient of 0.88. Et/Ald fixed cells incubated with distilled H2O had fewer granules containing Ca, 10%, than unincubated cells, 77%. Further, H2O removed all Ca from Et/Ald fixed cells embedded in DEO. These studies show that S, which is present as SO4 on the proteoglycan heparin, is readily detectable by X-ray EDS in fixed and embedded cells. An artifact of the technique is that weak anionic sites, which are most probably carboxyl groups on the proteoglycan, can bind the divalent cation Ca and cause spurious localization.

Graded dextrans have been used as tracers to identify the primary permeability barrier(s) to macromolecules among the structural elements (endothelium, mesangium, basement membrane, epithelium) of the glomerular capillary wall. Three narrow-range fractions of specified molecular weights and Einstein-Stokes radii (ESR) were prepared by gel filtration: (a) 32,000 mol wt, ESR = 38 Å; (b) 62,000 mol wt, ESR = 55 Å; and (c) 125,000 mol wt, ESR = 78 Å. These fractions are known to be extensively filtered, filtered in only small amounts, and largely retained, respectively, by the glomerular capillaries. Tracer solutions were infused i.v. into Wistar-Furth rats, and the left kidney was fixed after 5 min to 4 h. The preparations behaved as predicted: initially, all three fractions appeared in the urinary spaces, with 32,000 > 62,000 » 125,000. The smallest fraction was totally cleared from the blood and urinary spaces by 2.5 h, whereas the intermediate and largest fractions were retained in the circulation at high concentrations up to 4 h. With all fractions, when particles occurred in high concentration in the capillary lumina, they were present in similarly high concentrations in the endothelial fenestrae and inner (subendothelial) portions of the basement membrane, but there was a sharp drop in their concentration at this level—i.e., between the inner, looser portions of the basement membrane and its outer, more compact portions. With the two largest fractions, accumulation of particles occurred against the basement membrane in the mesangial regions with time. No accumulation was seen with any of the fractions in the epithelial slits or against the slit membranes. Dextran was also seen in phagosomes in mesangial cells, and in absorption droplets in the glomerular and proximal tubule epithelium. It is concluded that the basement membrane is the main glomerular permeability barrier to dextrans, and (since their behavior is known to be similar) to proteins of comparable dimensions (40,000–200,000 mol wt). The findings are discussed in relation to previous work using electron-opaque tracers to localize the glomerular permeability barrier and in relation to models proposed for the functions of the various glomerular structural elements.

Cercariae are covered by a thick glycocalyx (GCX) that activates complement by the alternative pathway and is recognized by antibodies in the serum of infected animals (Standen 1952; Samuelson and Caulfield 1985). Further, it contains antigens reactive with protective monoclonal antibodies produced in rats and mice (Grzych et al., 1982; Ham et al. 1984). Recently, Veira et al. (1986) found that cercarial transformation fluid, which contained GCX and other cercarial components, suppressed the in vitro proliferative response of human peripheral blood lymphocytes to both schistosome antigens and mitogens. Despite this evidence that GCX is immunologically relevant in schistosome infections, its effect as a primary immunogen has not been studied. We have recently characterized GCX prepared with sodium dodecyl sulfate (SDS) or guanidine hydrochloride (GuHCl) as solubilizing agents (Caulfield et al. 1987). Both preparations were rich in carbohydrate, 40% for GuHCl-GCX and 80% for SDS-GCX, with fucose as the major sugar. Here we injected mice with pooled fractions of either preparation of GCX, using phosphate-buffered saline (PBS), Maalox, or Bacille Calmette-Guerin (BCG) as carriers and/or adjuvants. We assessed the effect of immunization with GCX by comparing the numbers of adult worms recovered (worm burden) be mesenteric vein perfusion (Stirewah et al. 1951; Warren and Peters 1967) in GCXand sham-immunized mice 6 weeks postcercarial challenge. The number of adult worms recovered from mice was dramatically different between GuHCl-GCX and sham-immunized groups. As shown in Table I, animals injected with GuHCl-GCX in either PBS or Maalox had increased adult worm burdens in ah four experiments. The data were compared statistically using a stratified Wilcoxon’s test (Lagakos 1982; Table I). The statistical significance of the difference between the GuHCl-GCX-immunized and sham-immunized controls was 0.02 < P < 0.01. Thus, GuHCl-GCX when administered with PBS or Maalox significantly increased the number of surviving adult worms at 6 weeks postinfection when compared to shamimmunized controls. When BCG was administered with GuHCI-GCX, we found that the adult worm burdens were the same as in mice which received BCG alone. Mice immunized with SDS-GCX gave very different results from mice immunized with GuHCl-GCX. In two separate experiments with SDS-GCX, the adult worm burdens of SDS-GCX-immunized and shamimmunized controls were not significantly different when PBS or Maalox were used as adjuvants. Thus, immunization with SDS-GCX did not affect the number of surviving adult worms when compared to control-immunized mice. Several immunological mechanisms could explain the increase in numbers of adult worms recovered in mice immunized with GuHCl-GCX. Immunization with GuHCl-GCX may have suppressed cellular immunity, as suggested by Veira et al. (1986). Other studies have also demonstrated that schistosome materials suppressed the proliferative response of peripheral blood lymphocytes in vitro (Dessaint et al. 1977; Colley et al. 1979; Camus et al. 1981). Significantly, egg antigens, some of which cross-react with the cercarial surface (Ham et al. 1984), have been implicated in the suppression of the cell-mediated immune response (Boros et al. 1975; Colley 1975; Naggar and Colley 1982). Alternatively, GuHCl-GCX-immunized mice may have produced blocking antibodies which would bind to and protect invading larval parasites. Blocking antibodies to cercarial tegumental epitopes have been demonstrated in the rat model, both in vitro and in vivo (Grzych et al. 1982; Khahfe et al. 1986). Lastly, immunization with GuHCl-GCX may have induced a state of tolerance with respect to the carbohydrate antigens in GCX, analogous to that induced

Cercariae are covered by a glycocalyx that is highly antigenic. Here, we have examined the surface of miracidia for a similar structure. The miracidia are covered by epithelial plates and syncytial ridges. By transmission electron microscopy, the plates and ridges were covered by a 0.5-μm-thick glycocalyx composed of a mesh of 9- to 10-nm fibrils that were stained by ruthenium red delivered in the aldehydes or ferrocyanide-reduced osmium tetroxide. Rabbit antibodies prepared against phenol extracted and chromatographed cercarial glycocalyx were detected by immunoelectron microscopy with secondary antibodies conjugated to horseradish peroxidase. Reaction product bound to both the miracidial and cercarial glycocalyx. In addition, the outer leaflets of the cercarial tegumental membrane and membranes of the miracidial surface structures, including plates, ridges, terebratorium, and sensory papillae, had reaction product. Controls incubated with nonspecific rabbit serum had no reaction product. By indirect immunofluorescence, antibodies against the cercarial glycocalyx stained both plates and ridges. As the miracidia transformed to sporocysts, the glycocalyx remained associated with the plates as they were sloughed. These studies demonstrate that miracidia possess a glycocalyx similar in structure and antigenicity to the cercarial glycocalyx.

The effects of lysophosphatidylcholine (lysoPC) on human erythrocyte (RBC) ghost morphology, transmembrane protein and lipid lateral mobilities, and membrane lipid composition were studied in order to elucidate mechanisms by which lysoPC immobilizes ghost membrane components [Golan, D. E., Brown, C. S., Cianci, C. M. L., Furlong, S. T., & Caulfield, J. P. (1986) J. Cell Biol. 103, 819-828]. Under standardized conditions 1.0-1.5 micrograms/mL egg lysoPC lysed 50% of RBCs and induced, in some ghosts, the formation of large patches of wrinkled membrane. Patches exhibited complete immobilization of glycophorin and band 3 and partial immobilization of the phospholipid analogue fluorescein phosphatidylethanolamine (Fl-PE), whereas adjacent smooth membrane areas manifested only partial immobilization of proteins and no immobilization of Fl-PE. Supralytic concentrations of lysoPC induced both progressive, homogeneous wrinkling of RBC ghost membranes and concentration-dependent decreases in the lateral mobilities of glycophorin, band 3, and Fl-PE. Complete immobilization of glycophorin and band 3 occurred at 8.4 micrograms/mL lysoPC and of Fl-PE at 16.8 micrograms/mL lysoPC. Monopalmitoylphosphatidylcholine (MPPC), the major component of egg lysoPC, induced both membrane wrinkling and a concentration-dependent decrease in Fl-PE mobility, with complete immobilization at 10 micrograms/mL. Other synthetic lysoPCs did not completely immobilize Fl-PE, although some caused membrane wrinkling. MPPC was incorporated into ghost membranes with a linear dependence (r = 0.97) on MPPC concentration. Relative to total membrane lipid, the lysoPC mole fraction increased from 0.2 +/- 0.1% at 0 micrograms/mL MPPC to 25 +/- 2% at 16 micrograms/mL MPPC.(ABSTRACT TRUNCATED AT 250 WORDS)