Enhancement of mixed leukocyte reaction and cytotoxic antitumor responses by heparin

The immunomodulating effects of heparin and natural and synthetic heparinoids (which are now undergoing clinical trials for the treatment of AIDS) on cellular immunity (DNA synthesis and cytotoxic responses of mouse lymphocytes to allogeneic cells and histocompatible tumors) were studied. The results showed that (1) high and low m.w. heparin enhanced mouse antitumor and antiallogeneic cell responses in vitro; (2) other sulfated heparinoids did not have this enhancing activity and some of them (including dextran sulfate) totally suppressed generation of cytotoxic cells; (3) these immunomodulating activities of heparin and heparinoids did not correlate with their anticoagulant effects, degree of sulfation, and mitogenic activity; (4) heparin did not increase the production of IL-2 and did not enhance the action of IL-2 on the cells in MLC, heparin also had no effect on the growth-promoting activity of IL-2 on cloned cytotoxic T cells; (5) heparin had a synergistic enhancing effect with IL-1 on the generation of cytotoxic cells in MLC; and (6) heparin abolished endothelial cell growth factor-induced suppression of cytotoxic response. The latter two effects by themselves, however, could not fully explain the entire immunoenhancing activity of heparin. These results indicate that heparin and heparinoids have multiple effects on the immune system and that some of them can enhance, whereas others can suppress cell-mediated responses.     

Differential responses to UVR by bacterioplankton and phytoplankton from the surface and the base of the mixed layer

SUMMARY 1. We tested the influence of ultraviolet radiation (UVR) and shallow stratification on phytoplankton and bacterioplankton from the surface and the base of the mixed layer in two boreal lakes in north-western Ontario, Canada.
2. We measured phytoplankton biomass and production, bacterioplankton production and plankton respiration after transplantation under three solar radiation treatments: ambient radiation (Photosynthetically active radiation (PAR) + ultraviolet-A (UVA) + ultraviolet-B (UVB)), minus UVB (PAR + UVA) and PAR only. We repeated this experiment on three occasions in each lake during the summer.
3. Solar stress (measured as reduced growth and photoinhibition) was generally only found in the 'base phytoplankton' (i.e. originating from the base of the mixed layer). No inhibition of photosynthesis by UVB exposure was found in near-surface phytoplankton. On the other hand, production of near-surface bacterioplankton was reduced following a 4-h UVR exposure but had increased after a 48-h exposure to both UVA and UVB compared with the PAR only treatment.
4. Negative effects of UVR on phytoplankton and bacterioplankton were not ubiquitous. We emphasise the importance of conducting experiments repeatedly, particularly those which test the effects of UVR on different community assemblages from different lakes.     

Synthesis and biophysical properties of polymerized human serum albumin

The use of many plasma expanders (PEs) is often limited by undesirable side effects, such as red blood cell (RBC) aggregation (hydroxyethyl starch), nephrotoxicity (dextran), and extravasation (albumin). Despite its natural prevalence in the bloodstream, human serum albumin (HSA) can increase the risk of mortality when administered to patients with increased vascular permeability (i.e., patients suffering from burns, septic shock, and endothelial dysfunction). The harmful extravasation of HSA can be limited by polymerizing HSA to increase its molecular size. In this study, HSA was nonspecifically cross-linked with glutaraldehyde at different cross-link densities by varying the molar ratio of glutaraldehyde to HSA. The results of this study show that the weight-averaged molecular weight (MW), viscosity, and extent of RBC aggregation of polymerized HSA increases with increasing cross-link density, whereas the colloid osmotic pressure (COP) decreases with increasing cross-link density. Interestingly, circular dichroism measurements indicate that the secondary structure of HSA is unaffected by polymerization. Altogether, these results show that glutaraldehyde can effectively cross-link HSA to produce high MW polymers, yielding a novel series of potential PEs that exhibit low COP and high viscosity.     

Hemostatic effects of polymerized albumin particles bearing rGPIa/IIa in thrombocytopenic mice

The recombinant fragment of the platelet membrane glycoprotein Ia/IIa (rGPIa/IIa) was conjugated to the polymerized albumin particles (polyAlb) with the average diameter of 180 nm. The intravenous administration of rGPIa/IIa-polyAlb to thrombocytopenic mice ([platelet] = 2.1+/-0.3 x 10(5) particles/ microL) with three doses of ca. 2.4 x 10(10), 7.2 x 10(10), and 2.4 x 10(11)particles/kg, respectively, significantly reduced their bleeding time to 426+/-71, 378+/-101, and 337+/-46 s, respectively, whereas that of the control groups (PBS) was 730+/-198 s. The injection of rGPIa/IIa-polyAlb (2.4 x 10(11)particles/kg) was approximately equal to the effect of the injection of the mouse platelets at a dose of 2.0 x 10(10) particles/kg. It was confirmed that rGPIa/IIa-polyAlb had a recognition ability against collagen and could contribute to the hemostasis in the thrombocytopenic mice as a platelet substitute.     

Novel membranes and surface modification able to activate specific cellular responses

The design of new polymeric biomaterials together with new strategies to modify membrane surface are crucial to optimise cell-biomaterial interactions in vivo and in vitro biohybrid systems. In this study we report on the novel semipermeable membranes synthesised from a polymeric blend of modified polyetheretherketone and polyurethane able to support the long-term maintenance and differentiation of human liver cells and on the surface modification of polyethersulfone membranes by plasma polymerisation of acrylic acid monomers and by immobilization of arginine-glycine-aspartic acid (RGD) peptide through a hydrophilic "spacer arm" molecule. The performance of the modified and unmodified membranes was tested by evaluation of the liver function expression of primary human hepatocytes in terms of albumin production, protein secretion and drug biotransformation.     

Lymphocyte surface receptors and albumin

Albumin was shown to be hidden component of mouse B and T lymphocyte plasma membranes. It was readily radiolabeled from within the plasma membrane by a lipophilic, photoactivated reagent (125I-iodonaphthylazide) but not from the cell exterior by lactoperoxidase-catalyzed iodination; it was not detected by immunofluorescence on intact cells. The function of this cryptic lymphocyte membrane albumin is unknown at present. This cryptic albumin component was discovered during immunoprecipitation experiments using anti-Ig reagents. It is considered of general methodologic significance that many antisera, both native and rigorously Ig-absorbed (both positively and negatively), contained such contaminating activity. The possibility is raised that such contaminating activity may be involved in some reported findings of albumin-size "Ig-like heavy chains" in both the B and T lymphocyte lineages.     

Humoral and cellular responses to native antigen following oral and parenteral immunization with lipid-conjugated bovine serum albumin

Humoral and cellular immune responses of rabbits to bovine serum albumin (BSA) were measured following oral and parenteral immunization with either BSA or one of two dodecanoic acid conjugates of BSA. The first consisted of a mixture of lightly and heavily conjugated BSA-molecules (L-BSA-mix), while the second (L-BSA) was a homogeneous preparation of heavily conjugated BSA with more than 95% of the 60 available amino groups covalently bound to dodecanoic acid. Animals ingesting L-BSA-mix had a similar humoral immune response but enhanced cellular reactivity to BSA in comparison to animals ingesting the native antigen. No systemic immunologic responses to BSA were detected following ingestion of L-BSA in spite of the demonstration of circulating BSA antigenic groups. This lack of a detectable immune response after oral administration was not due to masking of antigenic sites by the lipid residues since both humoral and cellular immune responses to BSA were obtained in animals injected with L-BSA. Ingestion of L-BSA did not induce tolerance since a subsequent injection of BSA elicited a normal primary immune response. The differences in immunogenicity between BSA, L-BSA and L-BSA-mix following oral administration may be related to different modes of antigen recognition by the gut-associated lymphoid tissues.     

A simple model of plankton population dynamics coupled with a LES of the surface mixed layer

The concentration of phytoplankton in the sea is affected by biological processes, such as growth/mortality rates, predatory zooplankton concentrations and nutrient levels. Phytoplankton concentrations are also influenced by physical processes, in particular the mixing properties of the local fluid environment. On planktonic scales (approximately 10-1000 microm) one can assume the local turbulent flow is isotropic, with no distinction between horizontal and vertical mixing. However, agglomerations of phytoplankton into patches are observed on larger scales of up to hundreds of metres, whose formation will be influenced by the anisotropic advection/mixing properties and large-eddy structures prevalent in the surface mixed layer. This paper presents the results of the coupling of a large-eddy simulation (LES) model of the mixed layer with an advection-diffusion system of coupled equations for nitrate-phytoplankton-zooplankton (NPZ) concentration, incorporating sub-grid parameterizations of the biological processes. Typically these include phytoplankton growth due to light levels and ambient nitrate concentration, offset by grazing losses due to the presence of zooplankton. The primary goal of this work is to investigate how the characteristics of the mixed layer turbulence influence the observed distribution of phytoplankton. One novel feature is the incorporation of a 'vortex-force' term in the LES code in order to generate Langmuir circulations. It has been speculated that the enhanced mixing rates associated with 'Langmuir turbulence' play a significant role in regulating planktonic activity. Results derived from the coupled LES-NPZ model, run with and without the presence of Langmuir circulations, are presented in order to investigate these ideas.     

Generic signal-specific responses: cytokinin and context-dependent cellular responses

The phytohormone cytokinin triggers numerous and diverse responses during the plant life cycle via a two-component phosphorelay signalling system. Each step of the signalling cascade is supported by a gene family comprising several members. While functional redundancy is observed among family members, additional gene-specific functions encoded by cis-regulatory and coding sequence of individual family members have been described and contribute to specificity in signalling output. In addition, the cellular context of the signal-receiving cell affects the response triggered. Recent studies in Arabidopsis have demonstrated how expression of cytokinin signalling components predefines a spatiotemporal map of signalling sensitivity, which causes local signal amplification and attenuation. In summary, the specific interpretation of cytokinin signalling is affected by an orchestrated interplay of signalling genes and cellular context.     

Interaction between various polymerized human albumins and hepatitis B surface antigen.

A variety of albumin polymers were prepared and tested for binding with hepatitis B surface antigen (HBsAg): synthetic polymers cross-linked by either glutaraldehyde or carbodiimide; heat-aggregated polymers made by heating albumin solutions at 60 degrees C for 10 h with or without albumin stabilizer; and polymers isolated from fresh or long-stored commercial therapeutic albumin solutions. A sensitive solid-phase, competitive-inhibition radioimmunoassay, which can detect as little as 10 ng of glutaraldehyde-cross-linked human albumin polymer (PHALB-G), was developed and used to measure binding. The binding of PHALB-G with HBsAg was 150- to 1,000-fold greater than that of any other albumin polymer. Glutaraldehyde-cross-linked bovine albumin polymer showed no binding. Albumin monomer and dimer fractions produced by glutaraldehyde treatment exhibited some binding, albeit much weaker than PHALB-G. As measured by a direct-binding assay with solid-phase PHALB-G, the attachment of HBsAg particles from sera positive for antibody to the e antigen was less efficient than that from sera positive for e antigen, even when all sera were tested at equal HBsAg concentrations. In protein blot experiments with radiolabeled albumin preparations, PHALB-G bound almost exclusively to HBsAg polypeptide P31 and showed no binding with the major polypeptides P23 and P26. None of the other radiolabeled albumin polymers was reactive. These results indicate that the interaction between PHALB-G and HBsAg is not due to polymerization of albumin per se, but rather is unique and site specific.     

Glycobiology of surface layer proteins

Over the last two decades, a significant change of perception has taken place regarding prokaryotic glycoproteins. For many years, protein glycosylation was assumed to be limited to eukaryotes; but now, a wealth of information on structure, function, biosynthesis and molecular biology of prokaryotic glycoproteins has accumulated, with surface layer (S-layer) glycoproteins being one of the best studied examples. With the designation of Archaea as a second prokaryotic domain of life, the occurrence of glycosylated S-layer proteins had been considered a taxonomic criterion for differentiation between Bacteria and Archaea. Extensive structural investigations, however, have demonstrated that S-layer glycoproteins are present in both domains. Among Gram-positive bacteria, S-layer glycoproteins have been identified only in bacilli. In Gram-negative organisms, their presence is still not fully investigated; presently, there is no indication for their existence in this class of bacteria. Extensive biochemical studies of the S-layer glycoprotein from Halobacterium halobium have, at least in part, unravelled the glycosylation pathway in Archaea; molecular biological analyses of these pathways have not been performed, so far. Significant observations concern the occurrence of unusual linkage regions both in archaeal and bacterial S-layer glycoproteins. Regarding S-layer glycoproteins of bacteria, first genetic data have shed some light into the molecular organization of the glycosylation machinery in this domain. In addition to basic S-layer glycoprotein research, the biotechnological application potential of these molecules has been explored. With the development of straightforward molecular biological methods, fascinating possibilities for the expression of prokaryotic glycoproteins will become available. S-layer glycoprotein research has opened up opportunities for the production of recombinant glycosylation enzymes and tailor-made S-layer glycoproteins in large quantities, which are commercially not yet available. These bacterial systems may provide economic technologies for the production of biotechnologically and medically important glycan structures in the future.     

Covalent immobilization of chitosan and heparin on PLGA surface

Chitosan and heparin were covalently immobilized onto a poly(lactic acid-co-glycolic acid) (PLGA) surface using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS) in a 2-morpholinoethane sulfonic acid (MES) buffer system. The properties of the modified PLGA surface and the control were investigated by water contact angle measurement and electron spectroscopy for chemical analysis (ESCA). The water contact angle of the modified film was greatly decreased and the element content on the surface of the films changed correspondingly. Platelet adhesion assay showed that blood compatibility of the chitosan/heparin modified film was improved while hepatocyte culture indicated that the cell compatibility of the modified film was enhanced.     

Ceramide and other sphingolipids in cellular responses

Formerly considered to serve only as structural components, sphingolipids are emerging as an important group of signaling molecules involved in many cellular events, including cell growth, senescence, meiotic maturation, and cell death. They are also implicated in functions such as inflammation and the responses to heat shock and genotoxic stress. Defects in the metabolism of sphingolipids are related to various genetic disorders, and sphingolipids have the potential to serve as therapeutic agents for human diseases such as colon cancer and viral or bacterial infections. The best-studied member of this family, ceramide, which also serves as the structural back-bone for other sphingolipids, is an important mediator in multiple cellular signaling pathways. The metabolism and functions of sphingolipids are discussed in this review, with a focus on ceramide regulation in various cellular responses.     

Motion of polymerized albumin particles in a model arteriole in the presence of red blood cells

Polymerized albumin particles (poly Alb) with recombinant glycoprotein Ibα (rGPIbα-poly Alb) are a promising candidate for a platelet substitute. Thus, we focused on the lateral motion of poly Alb in the presence of red blood cells, because the lateral motion plays an important role in aggregate formation. We visualized the microscopic motion of poly Alb toward the immobilized ligand (von Willebrand factor, VWF) surface in a model arteriole with red blood cells with a high-speed camera. At a higher shear rate of 1,500 s⁻¹, the concentration profile of poly Alb appeared to peak near the wall. This profile enhances the interaction between the particles and wall. Particularly the migration angle, being the angle of the poly Alb velocity vector, was enlarged near the wall and contributed to transfer of poly Alb toward the immobilized VWF surface. This tendency is desirable to achieve the adhesion of particles on the wall.     

CD39 and control of cellular immune responses

CD39 is the cell surface-located prototypic member of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) family. Biological actions of CD39 are a consequence (at least in part) of the regulated phosphohydrolytic activity on extracellular nucleotides. This ecto-enzymatic cascade in tandem with CD73 (ecto-5–nucleotidase) also generates adenosine and has major effects on both P2 and adenosine receptor signalling. Despite the early recognition of CD39 as a B lymphocyte activation marker, little is known of the role of CD39 in humoral or cellular immune responses. There is preliminary evidence to suggest that CD39 may impact upon antibody affinity maturation. Pericellular nucleotide/nucleoside fluxes caused by dendritic cell expressed CD39 are also involved in the recruitment, activation and polarization of naïve T cells. We have recently explored the patterns of CD39 expression and the functional role of this ecto-nucleotidase within quiescent and activated T cell subsets. Our data indicate that CD39, together with CD73, efficiently distinguishes T regulatory cells (Treg) from other resting or activated T cells in mice (and humans). Furthermore, CD39 serves as an integral component of the suppressive machinery of Treg, acting, at least in part, through the modulation of pericellular levels of adenosine. We have also shown that the coordinated regulation of CD39/CD73 expression and of the adenosine receptor A2A activates an immunoinhibitory loop that differentially regulates Th1 and Th2 responses. The in vivo relevance of this network is manifest in the phenotype of Cd39-null mice that spontaneously develop features of autoimmune diseases associated with Th1 immune deviation. These data indicate the potential of CD39 and modulated purinergic signalling in the co-ordination of immunoregulatory functions of dendritic and Treg cells. Our findings also suggest novel therapeutic strategies for immune-mediated diseases.     

Regulation of integrin-mediated cellular responses through assembly of a CAS/Crk scaffold

The molecular coupling of CAS and Crk in response to integrin activation is an evolutionary conserved signaling module that controls cell proliferation, survival and migration. However, when deregulated, CAS/Crk signaling also contributes to cancer progression and developmental defects in humans. Here we highlight recent advances in our understanding of how CAS/Crk complexes assemble in cells to modulate the actin cytoskeleton, and the molecular mechanisms that regulate this process. We discuss in detail the spatiotemporal dynamics of CAS/Crk assembly and how this scaffold recruits specific effector proteins that couple integrin signaling networks to the migration machinery of cells. We also highlight the importance of CAS/Crk signaling in the dual regulation of cell migration and survival mechanisms that operate in invasive cells during development and pathological conditions associated with cancer metastasis.     

Shape and surface properties of titanate nanomaterials influence differential cellular uptake behavior and biological responses in THP-1 cells

We investigated cellular uptake behavior and biological responses of spherical and fibrous titanate nanomaterials in human monocyte THP-1 cells. Two titanate nanofibers (TiNFs), namely TF-1 and TF-2, were synthesized from anatase TiO₂ nanoparticles (TNPs) via hydrothermal treatment. The synthesized TiNFs and TNPs were thoroughly characterized for their size, crystallinity, surface area and surface pH. TF-1 (～2 µm in length) was amorphous with an acidic surface, while TF-2 (～7 µm in length) was brookite with a basic surface. The results demonstrated that none of these titanate nanomaterials resulted in significant cytotoxicity, even at the highest doses tested (50 µg/ml), consistent with an absence of ROS generation and lack of change of mitochondrial membrane potential. While no cytotoxic effect was found in the titanate nanomaterials, TF-2 tended to decrease the proliferation of THP-1 cells. Furthermore, TF-2 resulted in an inflammatory cytokine response, as evidenced by dramatic induction of IL-8 and TNF-α release in TF2 but not TF-1 nor TNPs. These results suggest that shape of titanate nanomaterials plays an important role in cellular internalization, while surface pH may play a prominent role in inflammatory response in THP-1 cells.