Specific binding of concanavalin A to free inositol and liposomes containing phosphatidylinositol

We previously reported that concanavalin A could bind specifically to liposomes containing phospholipids and lacking glycoconjugates (Biochem. Biophys. Res. Comm. 74, 208, 1977). In the present study we show that the binding of concanavalin A to the liposomes was greatly increased (up to 5 fold) by the presence of phosphatidylinositol in the liposomes. Furthermore, the binding of concanavalin A to phosphatidylinositol-liposomes was specific and could be inhibited by either alpha-methyl mannoside or by myo-inositol. We also found that concanavalin A-induced lymphocyte mitogenesis could be inhibited either by alpha-methyl mannoside or by myo-inositol. Simultaneous addition of both inhibitors to concanavalin A and liposomes showed that inhibition was non-competitive: alpha-methyl mannoside was more inhibitory to liposomes lacking phosphatidylinositol, and myo-inositol was more inhibitory to liposomes containing phosphatidylinositol. This suggests that the binding site for inositol might be different than that for mannose. Equilibrium dialysis and Scatchard plots revealed 4 binding sites each for inositol and mannose at neutral pH. The binding constants of concanavalin A were 0.13 X 10(4) and 0.25 X 10(4) liters/mole respectively for inositol and mannose. We conclude that concanavalin A binds specifically to the inositol portion of phosphatidylinositol.     

Heterogeneity of lipid A: comparison of lipid A types from different gram-negative bacteria

Chloroform-soluble purified lipid A preparations from 10 sources, including five Escherichia coli strains (EH100, K-12, O127, O111, RCDC), two Salmonella strains (Salmonella typhimurium, Salmonella minnesota R595), Shigella sonnei II, and a hybrid of Shigella flexneri and E. coli K-12, were compared with lipid A from S. flexneri. Purified lipid A from S. flexneri was earlier found to be composed of eight fractions. The various lipid A preparations were assayed by thin-layer chromatography. Chromatograms were stained for phosphate or carbohydrate by molybdenum blue or orcinol, respectively. The number of major bands found for each lipid A preparation varied between 7 and 10, depending on the source. Comparable bands, based on Rf, were found among all of the different lipid A preparations, but the quantity of each band varied between the sources of lipid A. Four bands (designated 2, 3, 7, and 8) were abundant in every preparation. Variations of conditions used for preparing lipid A, such as changing of hydrolysis time, did not affect the appearance of lipid A on thin-layer chromatography. Change in the type of acid used for hydrolysis also did not affect the band pattern, but it did change the quantitative amounts of the various bands to some degree.     

Membrane lipid composition modulates the binding specificity of a monoclonal antibody against liposomes

A hybridoma secreting a monoclonal IgM 'anti-liposome' antibody was produced after injecting a mouse with liposomes containing dipalmitoylphosphatidylcholine, cholesterol, dicetyl phosphate, and lipid A. The antibody was selected by assaying for complement-dependent damage to liposomes lacking lipid A. The monoclonal antibody reacted best with liposomes containing the original immunizing mixture of lipids. Deletion of individual lipid constituents from liposomes diminished the ability of the liposomes to bind (adsorb) the antibody. Binding of the antibody was enhanced by including lipid A or galactosylceramide in the lipid bilayer, or by substituting egg phosphatidylcholine for dimyristoyl- (or dipalmitoyl-) phosphatidylcholine. Sphingomyelin could be substituted for dimyristoylphosphatidylcholine without altering the adsorption of antibody. Although the monoclonal anti-liposome antibody was completely inhibited by phosphocholine, it was probably not a conventional anti-phosphocholine antibody. The antibody apparently had a partial specificity for phosphate, and was inhibited by glycerophosphocholine, glycerophosphate, sodium phosphate, sodium sulfate, and inositol hexaphosphate, but not by choline or inositol.     

Vitamin A in liposomes. Inhibition of complement binding and alteration of membrane structure.

Incorporation of vitamin A aldehyde (retinal) into liposomes had an inhibitory effect on the amount of human complement protein bound in the presence of specific antiserum. The total membrane-bound protein was directly measured on liposomes which were washed after incubation in antiserum and fresh human serum (complement). At every concentration of complement, decreased protein binding was found with liposomes which contained retinal. Binding of the third component of complement (C3) was also measured directly on washed liposomes and was found to be decreased in the presence of retinal. The diminution in protein binding due to retinal was not caused by differences in the amount of antibody bound and this was shown by two experiments. First, specific antibody protein binding to liposomes was directly measured and was essentially unaffected by retinal. Second, liposomes were prepared from lipid extracts of sheep erythrocytes. These liposomes were used as as immunoadsorbants to remove antisheep erythrocyte antibodies. The immunoadsorbant capacity was the same in both the presence and the absence of retinal. A further conclusion from these experiments was that retinal did not change the number of liposomal glycolipid antigen molecules available for antibody binding and thus presumably did not change the total number of lipid molecules present on the outer surface of the liposomes. Retinal did have an effect on the geometric structure of the liposomes. Size distribution measurements were performed in the diameter range of 1-6.35 mum by using an electronic particle size analyzer (Coulter Counter). Liposomes containing retinal were shifted toward smaller sizes and had less total surface area and volume. It was suggested that retinal-containing liposomes may have had a tighter packing of the molecules in the phospholipid bilayer. This effect of retinal on liposomal structure may have been responsible for the observed decreased binding of C3 and total complement protein.     

Immunohistochemistry in the evaluation of neovascularization in tumor xenografts

Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry is widely used in experimental studies for quantifying tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected by variations in the methodology used to measure vascularization including antibody selection, antigen retrieval (AR) pretreatment, and evaluation techniques. To examine this further, we investigated the microvessel density (MVD) and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg immunohistochemical staining. Different AR methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, anti-CD31 highlighted small vessels better than F VIII RAg. Furthermore, the MVD of CD31 was significantly greater than that of F VIII RAg decorated vessels (p<0.001). The choice of antibody and AR method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.     

Platelets and Erythrocyte-Bound Platelets Bind Infectious HIV-1 in Plasma of Chronically Infected Patients

Chronic HIV-1 infection is associated with persistent viremia in most patients, but it remains unclear how free virus may survive the potential hostile effects of plasma. We investigated whether sites might exist on the surfaces of circulating blood cells for protection of infectious HIV-1 particles. Red blood cells (RBC) either from blood of uninfected normal individuals, or from blood obtained without EDTA from chronically infected HIV-1 patients, invariably contained a small number of RBC having attached platelets as determined by flow cytometry, light microscopy, and immunofluorescence microscopy. After mixing normal RBC with platelet-rich plasma, discrete populations of RBC, platelets, and complexes of platelets attached to RBC were purified by fluorescence-activated cell sorting. Upon incubation of purified cells or platelets with HIV-1 followed by washing and co-incubation with CD4-positive peripheral blood mononuclear cells (PBMC), platelets, and platelet-RBC complexes, but not platelet-free RBC, caused infection of PBMC. Infection was prevented by pre-treating the platelet-RBC complexes with EDTA. Plasma and RBC (comprising a RBC/platelet-RBC mixture) from chronically infected patients with low viral loads were also co-incubated with PBMC ex vivo to determine the presence of infectious HIV-1. All freshly isolated plasmas from the HIV-1-infected donors, obtained in the absence of anticoagulant, were noninfectious. Interestingly, the RBC from most of the patients caused cell-cell infection of PBMC that was prevented by stripping the RBC with EDTA. A monoclonal antibody to DC-SIGN partially inhibited cell-cell HIV-1 infection of PBMC by normal RBC pre-incubated with platelets and HIV-1. We conclude: (a) platelet-free EDTA-free plasma from chronically infected HIV-1 patients, although containing viral RNA, is an environment that lacks detectable infectious HIV-1; (b) platelets and platelet-RBC complexes, but not purified RBC, bind infectious HIV-1; (c) DC-SIGN, and possibly other C-type lectins, may represent binding sites for infectious HIV-1 on platelets and platelet-RBC complexes.     

Saturated phospholipids are required for nano- to micron-size transformation of cholesterol-containing liposomes upon QS21 addition

Abstract

Liposomes containing cholesterol and monophosphoryl lipid A (such as ALFQ and AS01B) are vaccine adjuvants. During construction of the formulations, addition of QS21 to nano-size (50–100?nm) liposomes resulted in extremely large (up to ～30 µm) liposomes in ALFQ, but AS01B liposomes remained small nano-vesicles. Here, we show that saturation of phospholipid chains is essential for production of large liposomes by QS21.     

Designing a Soluble Near Full-length HIV-1 gp41 Trimer

The HIV-1 envelope spike is a trimer of heterodimers composed of an external glycoprotein gp120 and a transmembrane glycoprotein gp41. gp120 initiates virus entry by binding to host receptors, whereas gp41 mediates fusion between viral and host membranes. Although the basic pathway of HIV-1 entry has been extensively studied, the detailed mechanism is still poorly understood. Design of gp41 recombinants that mimic key intermediates is essential to elucidate the mechanism as well as to develop potent therapeutics and vaccines. Here, using molecular genetics and biochemical approaches, a series of hypotheses was tested to overcome the extreme hydrophobicity of HIV-1 gp41 and design a soluble near full-length gp41 trimer. The two long heptad repeat helices HR1 and HR2 of gp41 ectodomain were mutated to disrupt intramolecular HR1-HR2 interactions but not intermolecular HR1-HR1 interactions. This resulted in reduced aggregation and improved solubility. Attachment of a 27-amino acid foldon at the C terminus and slow refolding channeled gp41 into trimers. The trimers appear to be stabilized in a prehairpin-like structure, as evident from binding of a HR2 peptide to exposed HR1 grooves, lack of binding to hexa-helical bundle-specific NC-1 mAb, and inhibition of virus neutralization by broadly neutralizing antibodies 2F5 and 4E10. Fusion to T4 small outer capsid protein, Soc, allowed display of gp41 trimers on the phage nanoparticle. These approaches for the first time led to the design of a soluble gp41 trimer containing both the fusion peptide and the cytoplasmic domain, providing insights into the mechanism of entry and development of gp41-based HIV-1 vaccines.