Multi-functional Liposomes Enhancing Target and Antibacterial Immunity for Antimicrobial and Anti-Biofilm Against Methicillin-Resistant Staphylococcus aureus.

PURPOSE: The aim of this study was to prepare wheat germ agglutinin (WGA)-modified liposomes encapsulating clarithromycin and to evaluate their in vitro and in vivo efficacy against Methicillin-resistant Staphylococcus aureus (MRSA). METHODS: Physicochemical parameters, minimum inhibitory concentrations, in vitro killing kinetic, cellular uptake, biofilm formation inhibition and pre-formed biofilm destruction, biodistribution, in vivo antibacterial efficacy against MRSA, and phagocytosis into macrophages for liposomes loading clarithromycin were determined. RESULTS: The minimum inhibitory concentration and the time-kill curve for WGA-modified liposomal clarithromycin were better than those of free and nonmodified liposomal clarithromycin. Flow cytometry analysis displayed that liposomes could deliver more Coumarin 6, a fluorescent probe, into bacteria because of the conjugation of WGA. Besides, WGA-modified liposomal clarithromycin inhibited formation of S. aureus (ATCC 29213) and MRSA biofiom, and prompted the biofilm disassembly at lower concentrations below MIC. Effective accumulation of liposomes was displayed in the enterocoelia of the mice because of WGA. The number of MRSA colony-forming units in the kidney and spleen in mice treated with WGA-modified liposomal clarithromycin was significantly lower than that treated with free and nonmodified clarithromycin (p < 0.05). Intracellular localization of MRSA occurred in a significantly higher proportion of macrophage exposed to WGA-modified liposomes compared to those exposed to nonmodified liposomes. CONCLUSIONS: Liposome modified by WGA is a promising formulation for bacteria targeted delivery and immunity defensive system through macrophage improving uptake of bacteria, biodistribution, in vitro and in vivo antibacterial efficacy against MRSA.

The airway antigen sampling system: respiratory M cells as an alternative gateway for inhaled antigens.

In this study, we demonstrated a new airway Ag sampling site by analyzing tissue sections of the murine nasal passages. We revealed the presence of respiratory M cells, which had the ability to take up OVA and recombinant Salmonella typhimurium expressing GFP, in the turbinates covered with single-layer epithelium. These M cells were also capable of taking up respiratory pathogen group A Streptococcus after nasal challenge. Inhibitor of DNA binding/differentiation 2 (Id2)-deficient mice, which are deficient in lymphoid tissues, including nasopharynx-associated lymphoid tissue, had a similar frequency of M cell clusters in their nasal epithelia to that of their littermates, Id2(+/-) mice. The titers of Ag-specific Abs were as high in Id2(-/-) mice as in Id2(+/-) mice after nasal immunization with recombinant Salmonella-ToxC or group A Streptococcus, indicating that respiratory M cells were capable of sampling inhaled bacterial Ag to initiate an Ag-specific immune response. Taken together, these findings suggest that respiratory M cells act as a nasopharynx-associated lymphoid tissue-independent alternative gateway for Ag sampling and subsequent induction of Ag-specific immune responses in the upper respiratory tract.

In vivo toxicity and immunogenicity of wheat germ agglutinin conjugated poly(ethylene glycol)-poly(lactic acid) nanoparticles for intranasal delivery to the brain.

Biodegradable polymer-based nanoparticles have been widely studied to deliver therapeutic agents to the brain after intranasal administration. However, knowledge as to the side effects of nanoparticle delivery system to the brain is limited. The aim of this study was to investigate the in vivo toxicity and immunogenicity of wheat germ agglutinin (WGA) conjugated poly(ethylene glycol)-poly(lactic acid) nanoparticles (WGA-NP) after intranasal instillation. Sprague-Dawley rats were intranasally given WGA-NP for 7 continuous days. Amino acid neurotransmitters, lactate dehydrogenase (LDH) activity, reduced glutathione (GSH), acetylcholine, acetylcholinesterase activity, tumor necrosis factor α (TNF-α) and interleukin-8 (IL-8) in rat olfactory bulb (OB) and brain were measured to estimate the in vivo toxicity of WGA-NP. Balb/C mice were intranasally immunized by WGA-NP and then WGA-specific antibodies in serum and nasal wash were detected by indirect ELISA. WGA-NP showed slight toxicity to brain tissue, as evidenced by increased glutamate level in rat brain and enhanced LDH activity in rat OB. No significant changes in acetylcholine level, acetylcholinesterase activity, GSH level, TNF-α level and IL-8 level were observed in rat OB and brain for the WGA-NP group. WGA-specific antibodies in mice serum and nasal wash were not increased after two intranasal immunizations of WGA-NP. These results demonstrate that WGA-NP is a safe carrier system for intranasal delivery of therapeutic agents to the brain.

Tandem affinity depletion: a combination of affinity fractionation and immunoaffinity depletion allows the detection of low-abundance components in the complex proteomes of body fluids.

Protein biomarker discovery in the low concentration range of human body fluids requires the enrichment of the proteins of interest. Here we report on a tandem affinity strategy: In the first step, we isolated a human plasma glyco-subproteome of healthy individuals by wheat germ agglutinin (WGA) lectin affinity chromatography. In the second step, the proteins of this subproteome were used to raise antibodies in llama (Lama glama). The heavy-chain fraction of the llama antibodies was used to deplete from the WGA lectin binding fraction all proteins normally found in human plasma. In this way, we selectively enriched the glycoprotein, CEA, a known cancer marker which had been spiked into normal plasma. As a proof of concept, we applied this method to the analysis of plasma sample from colon cancer patients. We could demonstrate the selective enrichment of CEA by a factor of 600-800.

Rapid detection of ricin in cosmetics and elimination of artifacts associated with wheat lectin.

Ricin can be detected in cosmetics at 0.005 microg/mL in the analytical sample using lateral flow devices (LFDs). Wheat germ, an ingredient used in skin care products is also a potential source of wheat lectin. False positives were observed when wheat lectin was added to LFDs from two manufacturers, irrespective of whether the LFD was specific for ricin, Staphylococcus enterotoxin B (SEB), or botulinum toxin. In contrast, pea and peanut lectins did not cause false positives. Substitution of the buffer supplied with the LFDs with a buffer containing 2.5% non-fat milk powder eliminated the occurrence of false positives. This substitution increased the LOD to 0.01 microg/mL ricin, which is an acceptable level for screening cosmetics for contamination by ricin.

Mosquito midgut glycoproteins and recognition sites for malaria parasites.

Midgut glycoproteins of the malaria vector Anopheles tessellatus were partially characterised by gel electrophoresis and lectin binding. Specific binding to wheat germ agglutinin (WGA) and Concanavalin A (Con A) indicated the presence of N-linked core oligosaccharides in many proteins. Rabbit antibodies were produced against wheat germ agglutinin binding proteins (WGABP). These antibodies also recognised distinct proteins in the peritrophic membrane which is secreted into the midgut to enclose a bloodmeal. Rabbit anti-WGABP antibodies ingested in a bloodmeal containing infective gametocytes of the human malaria parasites Plasmodium falciparum and P. vivax tended to reduce infectivity of the parasites to vector mosquitoes. Chitotriose added to a bloodmeal also inhibited parasite development in the mosquito. The results are consistent with a hypothesis that N-acetyl glucosamine residues in mosquito midgut glycoproteins and/or midgut chitin and proteoglycan function as recognition sites for malaria parasites.

An immunoreactive protein to wheat-germ agglutinin antibody is induced in oat roots following invasion of the cereal cyst nematode Heterodera avenae, and by jasmonate.

A protein that cross-reacts to a wheat-germ agglutinin antibody was induced in oat roots following the invasion of second-stage juveniles (J2) of the cereal cyst nematode Heterodera avenae. This protein, designated ASP45, was acid soluble, and its molecular mass was about 45 kDa on a sodium dodecyl sulfate-polyacrylamide gel. ASP45 was induced in both compatible and incompatible interactions between the nematode and the plant, and also in roots by exposure to jasmonic acid (JA) or methyl jasmonate. However, ASP45 was not induced by elicitors of pathogenesis-related proteins, abscisic acid, or wounding. Lipoxygenase activity, which is involved in JA synthesis, was higher in nematode-infected and JA-treated roots than in their noninfected, untreated counterparts. Inhibition of lipoxygenase activity in roots abolished ASP45 induction in the nematode-infected roots. Amino acid sequences similar to that of ASP45 were found in chitinases of poplar tree and Arabidopsis, even though ASP45 showed no chitinase activity. Although the biological role of ASP45 in infected roots is not clear, JA is suggested to be involved in signal transduction after pathogen invasion of the plant.

Natural human antibodies to dietary lectins.

Natural antibodies to self and non-self proteins, including dietary proteins, are a significant part of the immune repertoire of humans. Antibodies to three structurally related legume lectins (Erythrina corallodendron lectin (ECorL), peanut agglutinin (PNA), and soybean agglutinin (SBA)) and to one cereal lectin (wheat germ agglutinin (WGA)) were purified by affinity chromatography from human sera and their binding specificity examined. The anti-SBA, anti-ECorL and anti-WGA antibodies exhibited high specificity, whereas the anti-PNA antibodies were polyreactive. Although the anti-WGA antibodies were highly specific for WGA, they also crossreacted slightly toward some other proteins. The anti-ECorL antibodies bound to native SBA, but the anti-SBA antibodies failed to bind to the native ECorL. Although the anti-SBA and anti-ECorL antibodies both exhibited specificity when interacting with native lectins, they bound to a wider range of denatured lectins, indicating a common or universal epitope which is recognized by many natural antibodies. Interestingly, the natural antibodies did not interfere with the agglutination properties of the lectins. These findings may provide a basis for studying the in vivo biological effects of anti-dietary protein antibodies, including those against carbohydrate-binding proteins.

Catecholaminergic afferents to the cat median eminence as determined by double-labelling methods.

The localization of dopaminergic and non-dopaminergic neuronal perikarya sending axons to the median eminence was investigated in the cat by using two colour double-immunostaining techniques. Unconjugated cholera toxin and wheat germ agglutinin were used as retrograde tracers and injected respectively into the median eminence and the neuro-intermediate pituitary of the same animal. As controls, cholera toxin was also injected into the arcuate (infundibular) nucleus or third ventricle. The retrograde labelling of one of the tracers was combined with tyrosine hydroxylase immunohistochemistry as a marker for dopaminergic neurons. The retrograde labelling studies of cholera toxin alone and the double-immunostaining of cholera toxin and wheat germ agglutinin on the same sections revealed that the cat median eminence receives major afferent projections originating in midline hypothalamic nuclear groups such as the anterior periventricular nucleus, the periventricular part of the paraventricular nucleus and the arcuate nucleus; minor afferent projections arise from the anterior hypothalamic area, the rostral part of the medial preoptic area around the organum vasculosum of the lamina terminalis and to a lesser extent from the posterior hypothalamic region. We further determine that the rostral part of the parvocellular arcuate neurons constitutes the main source of dopaminergic afferents to the median eminence in the cat brain.

Serum antibodies to wheat germ agglutinin and gluten in patients with dermatitis herpetiformis.

It has been speculated that gluten may play a role in the pathogenesis of dermatitis herpetiformis (DH) because it can act as a lectin. The lectin activity of gluten preparations was recently identified as wheat germ agglutinin (WGA). IgG and IgA serum antibodies to WGA and gluten were therefore measured in patients with DH and coeliac disease (CD) by an enzyme-linked immunosorbent assay (ELISA). Compared with healthy controls, both patients categories had increased IgG and IgA activities to WGA and gluten, the CD group showing the highest antibody levels. DH patients with subtotal villous atrophy tended to have higher activities than those with no villous changes or only minor changes. No significant difference in the gluten-to-WGA ratio of IgA or IgG antibodies was found when DH patients were compared with CD patients. If WGA plays a pathogenetic role in DH, then DH patients must have dermal characteristics, as yet undefined, that explain the initiation of their skin disease.

A specific wheat germ agglutinin-immunoreactive protein in human placenta.

In this study we examined human placenta for the presence of molecules antigenically related to a plant lectin, wheat germ agglutinin. The initial results of immunolocalization using polyclonal antibodies against wheat germ agglutinin showed that human placenta contains protein(s) recognized specifically. Staining of syncytiotrophoblast brush border and cytotrophoblast, granular in appearance was observed in first trimester human placenta. Specific binding was also seen in trophoblast-derived JAr and BeWo carcinoma cells. Isolation of wheat germ agglutinin-immunoreactive material from human placenta was achieved by ion-exchange- and affinity-chromatography on anti-wheat germ agglutinin-immunoglobulin G-Sepharose. The placental protein having molecular mass of 66 kD was identified as specific. The protein of 66 kD was characterized as a calcium-dependent, asialofetuin-binding molecule.

Immunological cross-reactivities between proteins secreted by the subcommissural organ, and plant lectins.

The glial subcommissural organ (SCO) discharges a glycoprotein-rich secretory product into the third ventricle to form Reissner's fibre (RF). The SCO proteins bear N-linked oligosaccharides, such as mediate many cell-cell and cell-matrix interactions. In such interactions the corresponding partner molecule recognising the sugar chain is often a sugar-specific protein (lectin). We present here evidence that the constituents of the SCO secretory product include proteins immunologically cross-reactive with certain plant lectins. Polyclonal antisera directed against Phaseolus vulgaris agglutinin-L (PHA-L) labelled the apically released RF-material of the rat SCO. Indirect ELISA studies shows in addition that anti-RF antisera bound to certain plant lectins (PHA-L, Con A). Dot spot assays demonstrated binding of anti-PHA-L to RF proteins. In Western blots of RF proteins anti-PHA-L, anti-RCA and anti-Con A bound to distinctive subsets of the RF protein fractions.

Immunoactivation of lectin-conjugated praecoxin A on IL-6, IL-12 expression.

Lectin-conjugated praecoxin A is a compound, which is combined Wheat Germ Agglutinin (WGA) Lectin with praecoxin A and also known to have an anti-tumor activity. In our lab, in order to investigate its immune reaction other than the anti-tumor activity ever known, we examined cytokines such as IL-6 and IL-12 through their mRNA expressions, which are generally secreted by macrophage both in vivo and in vitro. To analyze, we used RT-PCR for total RNAs of macrophages. As a result, we obtained that both in vitro and in vivo, lectin-conjugated praecoxin A showed an interesting increase on IL-6 and IL-12 even though it may be little hard to say the conjugated form is absolutely more effective than that of lectin or praecoxin A alone for immune response activities. Those results suggest that the conjugated form may give an additional opportunity in a future therapeutic use over its immuno activation properties.

Plasma membrane glycoproteins during spermatogenesis and in spermatozoa of some fishes.

Plasma membrane glycoproteins were analyzed during spermatogenesis and in the spermatozoa of the teleost fish Xiphophorus maculatus and of the Elasmobranch Schroederichthys chilensis. The analysis was undertaken using the fluoresceinated lectins concanavalin A (ConA) and wheat germ agglutinin (WGA) to detect glycoproteins in the plasma membrane using confocal laser microscopy. In Xiphophorus, a species with acrosomeless spermatozoa, primary spermatocytes and rounded shaped spermatids showed that the whole cell surface was labeled by both lectins. As spermiogenesis proceeded surface glycoproteins diminished and in mature spermatozoa a discrete and non-random localized fluorescence was observed exclusively on the surface of the spermatozoon head after the employ of ConA and WGA. In the elasmobranch Schroederichthys chilensis the spermatozoon displays an acrosome as a small vesicle. After ConA and WGA labeling, the region of the plasma membrane that covers the acrosome was the only fluorescent region in the gamete. The physiological significance of plasma membrane glycoproteins is discussed regarding spermatozoon physiology.

Synthesis, micellization and lectin binding of new glycosurfactants.

Here we report the preparation and physico-chemical characterization of carbohydrate-decorated micelles and their interaction with lectins. A library of biosourced amphiphiles was prepared by copper-catalyzed azide-alkyne cycloaddition (CuAAC) between alkynyl sugars (lactose, N-acetyl-D-glucosamine) and azido-functionalized poly(ethylene glycol) esters (N3-PEG900-decanoate (C10) and -dodecanoate (C12)). In water, these glycoconjugates self-assemble into micelles of homogeneous nanometric size (11 nm) as evidenced by scattering techniques (DLS for light, and SAXS for X-ray). A comparative study with previously synthesized octadecanoate counterparts pointed out that that nature of the fatty acid has no significant influence on the particle size but only affects their compactness. These findings are in favor of a possible bulk preparation from lipid mixtures such as those encountered in renewable vegetable oils. The presence of the carbohydrate epitopes on the surface of the micelles and their bioavailability for lectin targeting were also evidenced by light scattering measurements using wheat germ agglutinin (WGA) and peanut (Arachis hypogaea) (PNA) lectins, supporting possible application as targeted drug nanocarriers.

Overcoming soluble target interference in an anti-therapeutic antibody screening assay for an antibody-drug conjugate therapeutic.

BACKGROUND: The standard safety evaluation of biotherapeutics includes assessment of immunogenicity. Anti-therapeutic antibodies (ATA) can be detected in serum using immunoassays with a bridging format. However, these assays can be subject to interference. RESULTS: In the bridging ATA assay for 3A5 TDC, an antibody-drug conjugate that binds to the multimeric extracellular domain of MUC16 (CA125), soluble CA125 in the serum caused false-positive results by binding to the ATA assay reagents. This interaction was blocked by wheat germ agglutinin lectin as it binds to the glycans in CA125; thus, the specificity of the assay improved. CONCLUSION: The assay development and validation results showed that the addition of wheat germ agglutinin eliminates the interference from circulating CA125 without impacting the ability to detect ATA.

Lectins and the radioallergosorbent test.

An investigation into the possible role of lectin binding of IgE in RAST of legume and wheat extracts is reported. Lectins from pea, broad bean, lentil, jack bean, soybean, peanut, and wheat germ were coupled to RAST discs. The discs were pretreated with lectin-specific sugars in an attempt to inhibit RAST with sera from 11 sensitive patients. In all cases, RAST was almost unaffected by the inhibitory sugars, indicating that nonimmune binding of IgE by lectins in legume or wheat RAST was not significant when RAST was carried out with allergens bound to the usual paper discs. IgE contains binding sites for all the lectins examined, and five of the sera had high total IgE greater than 300 IU/ml. It is suggested that competition by IgG and other serum glycoproteins may explain the lack of effect by the added sugars. All sera contained endogenous glucose at a level of about 3 mmol/L that may have accounted for some self-inhibition of the lectin binding by pea, broad bean, lentil, and jack bean lectins. There was, however, significant immune binding of some of the lectins by specific IgE, and it is concluded that these lectins may be important in expression of IgE-mediated allergic responses.

Identification of human placental wheat germ agglutinin-immunoreactive protein by mass spectrometry.

A preparation of polyclonal antibodies to human placental calcium-dependent, carbohydrate-binding glycoprotein, previously identified as wheat germ agglutinin-immunoreactive protein, was applied as the ligand in an immunoaffinity procedure. Following electrophoretic separation of purified material, the specific 66-kDa antigen band was excised and subjected to in-gel protein cleavage. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of the tryptic digest yielded 42 isotopically resolved peptide fragments from mass 860 to 2,690. Empirical data from MALDI-MS were analyzed by computer assistance using ProteoMetrics PROFOUND software. Protein candidates reported in the specified identification database search are discussed in relation to the biochemical characteristics of the protein analyzed and its possible antigenic relatedness to wheat germ agglutinin. We speculate that a fibulin-like member of the epidermal growth factor-repeat protein family might be selected as a positive match.

Uptake of antitetanus F(ab')2 fragments into eukaryotic cells.

1. In order to introduce antitetanus immunoglobulin fragments into eukaryotic cells, either antitetanus F(ab')2 or Fab' fragments have been linked to carrier molecules. Aciclovir, horseradish peroxidase, wheat germ agglutinin, and transferrin were tried as carriers. 2. F(ab')2-aciclovir and Fab'-horseradish peroxidase were not internalized by NG108-15 neurohybridoma cells. 3. [Fab']2-wheat germ agglutinin and F(ab')2-transferrin conjugates were internalized into various cells. 4. F(ab')2-transferrin conjugates were made with three different linkers: N-succinimidyl 3-(2-pyridyldithio) propionate, bis-maleimido hexane, and bis-maleimidoethoxy propane. All three conjugates were internalized but had a different fate inside the cells.