Variable antibody-dependent activation of complement by functionalized phospholipid nanoparticle surfaces

A wide variety of nanomaterials are currently being developed for use in the detection and treatment of human diseases. However, there is no systematic way to measure and predict the action of such materials in biological contexts. Lipid-encapsulated nanoparticles (NPs) are a class of nanomaterials that includes the liposomes, the most widely used and clinically proven type of NPs. Liposomes can, however, activate the complement system, an important branch of innate immunity, resulting in undesirable consequences. Here, we describe the complement response to lipid-encapsulated NPs that are functionalized on the surface with various lipid-anchored gadolinium chelates. We developed a quantitative approach to examine the interaction of NPs with the complement system using in vitro assays and correlating these results with those obtained in an in vivo mouse model. Our results indicate that surface functionalization of NPs with certain chemical structures elicits swift complement activation that is initiated by a natural IgM antibody and propagated via the classical pathway. The intensity of the response is dependent on the chemical structures of the lipid-anchored chelates and not zeta potential effects alone. Moreover, the extent of complement activation may be tempered by complement inhibiting regulatory proteins that bind to the surface of NPs. These findings represent a step forward in the understanding of the interactions between nanomaterials and the host innate immune response and provide the basis for a systematic structure-activity relationship study to establish guidelines that are critical to the future development of biocompatible nanotherapeutics.     

Exploiting cholera vaccines as a versatile antigen delivery platform

The development of safe, immunogenic and protective cholera vaccine candidates makes possible their use as a versatile antigen delivery platform. Foreign antigens can be delivered to the immune system with cholera vaccines by expressing heterologous antigens in live attenuated vectors, as fusion proteins with cholera toxin subunits combined with inactivated Vibrio cholerae whole cells or by exposing them on the surface of V. cholerae ghosts. Progress in our understanding of the genes expressed by V. cholerae during infection creates unprecedented opportunities to develop an improved generation of vaccine vectors to induce immune protection against a broad range of pathogenic organisms.     

Absorption and lymphatic transport of cholesterol in the rat

Rats with thoracic duct fistulae were fed triolein and triolein containing various amounts of labeled cholesterol. The analysis of the lymph lipids gave the following results. In the fasting state the cholesterol transported via the thoracic duct was 0.87 micromole/hr. Feeding 800 micromoles of triolein gave a maximum rate of transport of cholesterol of 1.65 micromoles/hr. Addition of cholesterol to the triolein further increased the cholesterol transport to a maximal rate of almost 5 micromoles/hr when 50 micromoles of cholesterol were fed per 800 micromoles of triolein. The exogenous fraction of the cholesterol transported increased linearly with increasing cholesterol load, constituting at the highest dose almost 90% of the total cholesterol transported. An almost constant fraction (about 0.4) of the dietary cholesterol was recovered in the thoracic duct lymph in 24 hr irrespective of the dose fed, from a trace up to 100 micromoles in 800 micromoles of triolein. Cholesterol absorption has the characteristics of a passive diffusion process.     

Role of complement activation in atherosclerosis

PURPOSE OF REVIEW: Atherosclerosis is characterized by a strong inflammatory component. One factor contributing to inflammation in the arterial intima is the complement system. Here we summarize recent progress in the field of complement research on atherogenesis. RECENT FINDINGS: The complement system is activated in human atherosclerotic lesions and is actively regulated by the local synthesis of complement components and of complement regulatory proteins. Potential triggers of complement activation in the arterial intima include immunocomplexes, C-reactive protein, modified lipoproteins, apoptotic cells, and cholesterol crystals. Complement activation releases anaphylatoxins, and anaphylatoxin receptors have been identified in human atherosclerotic lesions. However, experiments on genetically engineered mice with severe hyperlipidemia have been unable to show a major role for complement in experimental atherogenesis. SUMMARY: In humans there is extensive circumstantial evidence for a role of complement in atherosclerosis, which is somewhat contradictory to recent modest or negative findings in atherosclerosis-prone genetically engineered hyperlipidemic mice.     

Exploiting virus-like particles as innovative vaccines against emerging viral infections

Emerging viruses pose a major threat to humans and livestock with global public health and economic burdens. Vaccination remains an effective tool to reduce this threat, and yet, the conventional cell culture often fails to produce sufficient vaccine dose. As an alternative to cell-culture based vaccine, virus-like particles (VLPs) are considered as a highpriority vaccine strategy against emerging viruses. VLPs represent highly ordered repetitive structures via macromolecular assemblies of viral proteins. The particulate nature allows efficient uptake into antigen presenting cells stimulating both innate and adaptive immune responses towards enhanced vaccine efficacy. Increasing research activity and translation opportunity necessitate the advances in the design of VLPs and new bioprocessing modalities for efficient and cost-effective production. Herein, we describe major achievements and challenges in this endeavor, with respect to designing strategies to harnessing the immunogenic potential, production platforms, downstream processes, and some exemplary cases in developing VLP-based vaccines.     

Absorption and lymphatic transport of cholesterol and sitosterol in the rat

An attempt was made to determine the mechanism for the greater absorbability of cholesterol as compared to sitosterol. Sitosterol-22,23-(3)H in different combinations with cholesterol-4-(14)C, dissolved in 0.8 ml of triolein, was fed to rats with lymph fistulae. Feeding 1.5, 50, or 100 micro moles of sitosterol resulted in a transfer to the lymph in 24 hr of 3-6% of the sitosterol, largely independent of the dose fed. The total amount of sitosterol transferred to the lymph was therefore almost linearly related to the dose fed. 30% of a tracer dose of cholesterol-4-(14)C fed together with the sitosterol was transferred to the lymph in 24 hr. When a total of 50 micro moles of sterol, containing cholesterol-(14)C and sitosterol-(3)H in the proportions 1:3, 1:1, and 3:1, was similarly fed, we found that sitosterol had no significant effect on the lymphatic transport of the simultaneously fed cholesterol. The ratio of (3)H to (14)C in the lymph was between 0.1 and 0.2 (the ratio in each fed mixture being taken as 1.0). The ratio was constant during the absorption period and independent of the ratio of sterols in the fed sterol mixture. Thus the same percentage of each sterol was always absorbed, and the sterols exerted no mutual interference in each others' absorption. We conclude that the mechanism for specificity in sterol absorption must be located early in the transport of the sterols within the intestinal mucosa cell.     

Systemic complement activation in age-related macular degeneration

Dysregulation of the alternative pathway (AP) of complement cascade has been implicated in the pathogenesis of age-related macular degeneration (AMD), the leading cause of blindness in the elderly. To further test the hypothesis that defective control of complement activation underlies AMD, parameters of complement activation in blood plasma were determined together with disease-associated genetic markers in AMD patients. Plasma concentrations of activation products C3d, Ba, C3a, C5a, SC5b-9, substrate proteins C3, C4, factor B and regulators factor H and factor D were quantified in patients (n = 112) and controls (n = 67). Subjects were analyzed for single nucleotide polymorphisms in factor H (CFH), factor B-C2 (BF-C2) and complement C3 (C3) genes which were previously found to be associated with AMD. All activation products, especially markers of chronic complement activation Ba and C3d (p<0.001), were significantly elevated in AMD patients compared to controls. Similar alterations were observed in factor D, but not in C3, C4 or factor H. Logistic regression analysis revealed better discriminative accuracy of a model that is based only on complement activation markers Ba, C3d and factor D compared to a model based on genetic markers of the complement system within our study population. In both the controls' and AMD patients' group, the protein markers of complement activation were correlated with CFH haplotypes.This study is the first to show systemic complement activation in AMD patients. This suggests that AMD is a systemic disease with local disease manifestation at the ageing macula. Furthermore, the data provide evidence for an association of systemic activation of the alternative complement pathway with genetic variants of CFH that were previously linked to AMD susceptibility.     

Systemic complement activation and acute lung injury

Experimental studies of rats have provided significant evidence that intravascular complement activation after i.v. injection of cobra venom factor (CVF) or thermal injury of skin can result in acute lung injury. This has been determined by morphological changes in lung and increases in lung vascular permeability. Systemic complement activation is associated with an early appearance of C5-derived chemotactic activity in the circulation coincident with the development of transient neutropenia, followed by extensive granulocytosis and sequestration of neutrophils in lung interstitial capillaries. The acute pulmonary injury depends on availability of complement and neutrophils. Depletion of either complement or blood neutrophils before CVF injection or thermal injury will prevent development of lung injury. Interventional studies with catalase, scavengers of hydroxyl radical OH., and iron chelators have revealed that the acute pulmonary injury is related to production of oxygen-derived free radicals by activated neutrophils. OH. appears to be the key mediator involved in the acute lung microvascular injury.     

Relative inefficiency of terminal complement activation

The efficiency of generation of fluid-phase SC5b-9 and membrane C5b-9(m) complexes relative to cleavage of C3 and C5 was studied. Fluid-phase C activation was induced through addition of purified bacterial Ag to human serum. Sephadex beads were used as particulate activators of the alternative pathway. Rabbit or antibody-coated sheep or human E were used to study formation of cytolytic C5b-9(m) complexes. The molar ratios of C3a:C5a generated in the model systems were found to be in the range of 60 to 200:1 in the case of soluble immune complex activators, and 70 to 150:1 with particulate activators and cells. The efficiency of C5 cleavage relative to C3 cleavage increased on surfaces with the density of antibody and/or C3b-binding sites. With soluble immune complexes, the efficiency of subsequent SC5b-9 generation displayed wide variations dependent on Ag and donor with molar ratios of C5a:SC5b-9 ranging from 30:1 for teichoic acid and sometimes approaching 1:1 for streptolysin-O. In contrast, activation on particles or cells always led to C5a:C5b-9 (calculated as the sum of generated moles SC5b-9 and C5b-9(m] ratios approaching 1:1. Hence, there is an overall inefficiency of terminal sequence activation in the C cascade due first to a dissociation at the level of C5 convertase formation/C5-cleavage and second, to a frequent inefficiency of C5b-utilization in the fluid-phase. The results provide an explanation for the very low levels of SC5b-9 found in plasma of healthy individuals and in patients with C-consuming immune complex disease.     

Exploiting natural immunity to helminth parasites for the development of veterinary vaccines

The development of subunit vaccines against most parasitic helminth infections will require a better understanding of the different components of a natural rejection process including (1) recognition of parasite antigens; (2) induction of protective immune response phenotypes; and (3) activation of appropriate immune effector mechanisms. While novel technologies have allowed significant progress to be made in the identification of candidate vaccine antigens, the large scale production of these antigens and their presentation to the host with appropriate adjuvant systems remains a major problem in vaccine research. Identification of the molecular interactions involved in the innate immune response to helminth infections and the application of new genomic and proteomic technologies are likely to lead to major advances in these research fields. Gastrointestinal nematode parasites and liver fluke are the most important helminth parasites of production animals. In recent years, a lot of new knowledge has been gathered on the immunobiology of the host-parasite interactions in these two infection systems, which has allowed new vaccination strategies to be considered. Functional genomic technologies such as gene expression analysis by microarrays, promise to further advance our understanding of the molecular pathways leading to protection against parasite infections. This will not only have implications for vaccine research, but also provide novel targets for drug development and genetic selection.     

Heparin and modified heparin inhibit complement activation in vivo.

Heparin regulates C activity in vitro, but has not been examined for this activity in vivo. The present study investigated the ability of commercial heparin and derivatized (N-desulfated, N-acetylated) heparin (Hep-NAc) with greatly diminished anticoagulant activity to inhibit C activation in guinea pigs. Catheters were placed in the right atrium of guinea pigs and kept patent with frequent saline flushes. The next day, heparin, Hep-NAc, or saline was given and 2.5 min later cobra venom factor or saline was given. Blood was drawn at intervals and assayed for total hemolytic C, C3 hemolytic activity, free hemoglobin, and activated partial thromboplastin time. Total hemolytic C and C3 activity decreased less rapidly in heparin- and Hep-NAc-pretreated animals than in non-pretreated animals, indicating that both heparins inhibited C activation. Heparin and Hep-NAc also inhibited cobra venom factor-induced hemolysis. This study demonstrates that commercial heparin and modified heparin inhibit C activation in vivo. This represents an important step in the development of an oligosaccharide drug to regulate C activation.     

Basic mechanisms controlling lymph transport in the mesenteric lymphatic net

This minireview summarizes an oral presentation given at the National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health workshop "Lymphatics in the Digestive System: Physiology, Health, and Disease" in Bethesda, Maryland on November 3-4, 2009. The concepts of extrinsic and intrinsic pumps, as well as intrinsic and extrinsic flows, are discussed in relation to the lymph transport in mesenteric lymphatic vessels. Age-related alterations in the structure and regulatory mechanisms of lymph flow in mesenteric lymphatic vessels may provide the basis for their diminished ability to work during the periods of increased functional loads in them. The recent development of modern experimental tools provides the opportunity to extend the knowledge on lymph transport function of lymphatic vessels that is absolutely necessary to maintain fluid and macromolecular homeostasis and to provide a transportation route for lipids adsorbed in gut and to immune cells.     

Transdiaphragmatic lymphatic transport of intraperitoneally administered marker in hamsters.

OBJECTIVE: To determine whether transdiaphragmatic transport in hamsters is similar to that described in other animals by examining transport of an intraperitoneally administered marker. METHODS: Monastral blue B suspension was administered intraperitoneally to 28 male Syrian hamsters (Mesocricetus auratus). Four hamsters each were euthanized 7, 15, and 30 min, and 1, 2, 3, and 24 h later. Specimens were examined microscopically for presence of marker. RESULTS: Marker was present in intrathoracic lymphatic vessels and cranial and caudal mediastinal lymph nodes by 7 min after its administration. The amount of marker in lymph nodes increased with time. The subcapsular distribution of marker was consistent with lymphatic transport. By 1 h after its administration, marker was present in the liver, spleen, bone marrow, and mesenteric and mandibular lymph nodes. Patterns of marker distribution in these tissues were consistent with hematogenous transport, but the amount of marker was considerably less than that in the intrathoracic lymph nodes at corresponding times. CONCLUSIONS: Particulates were most likely translocated from the hamster peritoneal cavity to intrathoracic lymph nodes via transdiaphragmatic lymphatic vessels. A portion of the translocated particulates entered the blood, where they were distributed to a variety of tissues within a short time.     

Temperature dependence of protein transport across lymphatic endothelium in vitro

The purpose of the work was to develop an in vitro model for the study of lymphatic endothelium and to determine, using this model, whether or not a cytoplasmic process may be involved in transendothelial transport. Segments of canine renal hilar lymphatics were dissected clean, cannulated at both ends, and transferred to a perfusion chamber for measurement of transendothelial protein transport and for ultrastructural tracer studies. The segments were subsequently processed for light and electron microscopy. By both structural and functional criteria the lymphatics were judged to have retained their integrity. At 37 degrees C, 36 lymphatics showed a mean rate of protein transport of 3.51 +/- 0.45 (SEM) micrograms/min per cm2 of lymphatic endothelium. The rate was influenced by the temperature of the system, being significantly reduced by 49% +/- 4.8, 31% +/- 5.3, and 29% +/- 3.9 when the temperature was lowered to 4 degrees, 24 degrees, and 30 degrees C, respectively. When the temperature was raised to 40 degrees C, the rate was significantly increased by 48% +/- 12.2. The vesicular system and the intercellular regions in vessels with increased or reduced rates of transport were analyzed quantitatively to ascertain whether the rate changes could be correlated with ultrastructurally demonstrable changes in either of these postulated pathways. No significant changes in junctional or vesicular parameters were found between the control lymphatics and those perfused at 24 degrees, 30 degrees, and 40 degrees C. At 4 degrees C, the temperature at which the rate of protein transport was maximally reduced, vesicular size decreased, and the number of free cytoplasmic vesicles increased, whereas the number associated with the abluminal and luminal surfaces decreased. We concluded that isolated perfused lymphatic segments transport protein at a relatively constant rate under control conditions, and that this transendothelial transport comprises both temperature-dependent and temperature-independent mechanisms. The findings were considered in terms of the different theories of lymph formation and were interpreted as providing support for the vesicular theory.     

Structure and function of lymphatic postcapillaries (coordinating mechanism of the processes of interstitial transport and lymphatic resorption

Investigation performed on topography, structure and resorptive function of lymphatic microvessels in the serous membranes gives possibilities to work out a model on formation and transport of lymph. Lymphatic postcapillaries (LP) - thin-walled endothelial channels with valves make an important link of the lymphatic system. The LP structure is similar to that of capillaries. These microvessels are situated in the interstitial space area, that are characterized with their high content of plasma proteins and water. Interrelation in concentrations of protein in the LP lumen and in the tissue ensures the existence of the osmotic gradient pressure through the wall and contributes liquor resorption into the lumen of microvessels. Peritoneal lymphatic capillaries are situated in the areas of a higher hydrotation level of the interstitial space. They can control the rate of the liquor filtration from plasma into tissue and regulate the resorption level in the whole lymphatic network. The model provides a differentiated participation of the LP and capillaries in performing resorption of proteins and liquor from the interstitium. The resorption mechanisms are closely connected with processes of the lymph movement along the vessels.     

Intestinal absorption and lymphatic transport of cholesterol and beta-sitostanol in the rat.

The intestinal absorption of cholesterol and beta-sitostanol (the saturated analogue of beta-sitosterol) were measured and their absorptions compared in the presence and absence of cholestyramine. After test meals containing [(3)H]cholesterol and [(14)C]beta-sitostanol without added cholestyramine, 4-day fecal collections yielded an average of 51% of the fed cholesterol and 83% of the fed beta-sitostanol. In separate lymph transport studies without cholestyramine, 36% of the fed cholesterol was recovered in lymph in 24 hours compared to only 2% of the fed beta-sitostanol. Thus, while total recoveries of the two labeled compounds in feces plus lymph were nearly identical (51% + 36% = 87% for cholesterol and 83% + 2% = 85% for beta-sitostanol) their distribution in the two compartments was markedly different, reflecting the relative nonabsorbability of beta-sitostanol. Adding cholestyramine to the test meal caused fecal excretion of cholesterol to increase to 73%, independent of the dose of cholestyramine used. Cholestyramine had no effect on the fecal excretion of beta-sitostanol (average excretion after cholestyramine, 85%). The relative non-absorbability of beta-sitostanol compared to cholesterol is clearly evident in this study and leads us to suggest its possible use as a lipid-soluble, nonabsorbable reference compound for measurement of the absorption of cholesterol and other lipids. Further data are presented to justify its use for this purpose.-Hassan, A. S., and A. J. Rampone. Intestinal absorption and lymphatic transport of cholesterol and beta-sitostanol in the rat.     

Surfactant protein A regulates complement activation.

Complement proteins aid in the recognition and clearance of pathogens from the body. C1, the first protein of the classical pathway of complement activation, is a calcium-dependent complex of one molecule of C1q and two molecules each of C1r and C1s, the serine proteases that cleave complement proteins. Upon binding of C1q to Ag-bound IgG or IgM, C1r and C1s are sequentially activated and initiate the classical pathway of complement. Because of structural and functional similarities between C1q and members of the collectin family of proteins, including pulmonary surfactant protein A (SP-A), we hypothesized that SP-A may interact with and regulate proteins of the complement system. Previously, SP-A was shown to bind to C1q, but the functional significance of this interaction has not been investigated. Binding studies confirmed that SP-A binds directly to C1q, but only weakly to intact C1. Further investigation revealed that the binding of SP-A to C1q prevents the association of C1q with C1r and C1s, and therefore the formation of the active C1 complex required for classical pathway activation. This finding suggests that SP-A may share a common binding site for C1r and C1s or Clq. SP-A also prevented C1q and C1 from binding to immune complexes. Furthermore, SP-A blocked the ability of C1q to restore classical pathway activity to C1q-depleted serum. SP-A may down-regulate complement activity through its association with C1q. We hypothesize that SP-A may serve a protective role in the lung by preventing C1q-mediated complement activation and inflammation along the delicate alveolar epithelium.