Probing the Origin of the Toxicity of Oligomeric Aggregates of α-Synuclein with Antibodies.

The aggregation of α-synuclein, a protein involved in neurotransmitter release at presynaptic terminals, is associated with a range of highly debilitating neurodegenerative conditions, most notably Parkinson's disease. Intraneuronal inclusion bodies, primarily composed of α-synuclein fibrils, are the major histopathological hallmarks of these disorders, although small oligomeric assemblies are believed to play a crucial role in neuronal impairment. We have probed the mechanism of neurotoxicity of α-synuclein oligomers isolated in vitro using antibodies targeting the N-terminal region of the protein and found that the presence of the antibody resulted in a substantial reduction of the damage induced by the aggregates when incubated with primary cortical neurons and neuroblastoma cells. We observed a similar behavior in vivo using a strain of C. elegans overexpressing α-synuclein, where the aggregation process itself is also partially inhibited as a result of incubation with the antibodies. The similar effects of the antibodies in reducing the toxicity of the aggregated species formed in vitro and in vivo provide evidence for a common origin of cellular impairment induced by α-synuclein aggregates.

Analysis of the oligomeric states of nucleophosmin using size exclusion chromatography.

Nucleophosmin (NPM1) is a multifunctional phosphoprotein which plays important roles in diverse biological processes. NPM1 can form homo- or hetero-oligomers through its N-terminal region, and bind DNA and RNA through its C-terminal region. However, the monomer-oligomer distribution of NPM1, and the extent of NPM1 binding and unbinding to RNA in living cells, are not fully understood. In this work, we analysed molecular complexes of NPM1 using size exclusion chromatography. We found that a substantial fraction of NPM1 behaves as an oligomer in HeLa cells. Furthermore, we identified three distinct oligomeric states of NPM1 using molecular characterization techniques such as subcellular localization and RNA binding. Finally, we found that heterozygous expression of a leukemia-associated NPM1 mutant significantly decreases the RNA binding level. Our data demonstrate that size exclusion chromatography provides a powerful tool for analysing NPM1 oligomers.

Linear α-(1 → 6)-d-glucan from Bifidobacterium bifidum BIM В-733D is low molecular mass biopolymer with unique immunochemical properties.

Role of microorganisms in induction of/protection from autoimmune diseases is proven though molecular mechanisms and bacterial/viral/yeast biopolymers responsible for these effects are in the research stage. Autoantobodies (AAbs) to thyroid peroxidase (anti-TPO) and thyroglobulin (anti-Tg) as well as AAbs to transglutaminase 2 (anti-TG2) and antibodies to gliadins (anti-gliadins) are serological markers of autoimmune thyroid disease and celiac disease, respectively, and players in pathogenesis of these autoimmune diseases. In current study, biopolymer of Bifidobacterium bifidum BIM В-733D that interacts selectively with anti-gliadins (Bb-Ganti-gliadins) was isolated by affinity chromatography with anti-gliadins, purified by size exclusion chromatography on TSK 40 gel and identified by NMR as linear α-(1 → 6)-d-glucan with molecular mass about 5000 Da. It was proven that compounds Bb-Ganti-gliadins and Bb-Ganti-TPO/Bb-Ganti-Tg isolated early from the same strain [Kiseleva, E. P. et al., Benef Microbes.2013, 4, 375 -391] are the same substance designated GBb. Its unique immunochemical property is the ability to interact selectively with anti-TPO, anti-Tg, anti-TG2 and anti-gliadins in presence of no less than 10-fold excess of total immunoglobulins of class G (tIgG), as it was proven by ELISA. Synthesis of GBb-bovine serum albumin (GBb-BSA) conjugate is an example of increasing the reliability and reproducibility of ELISA results by mediated immobilization of a polysaccharide covalently attached to a well-adsorbed protein. Taking into account that there are population of bispecific anti-gliadins (anti-gliadins and anti-TG2 simultaneously) we regard our data as first argument in favor of hypothesis that GBb differentiates between human AAbs per se and other human Ig (e.g. antibodies to antigens of infectious agents) due to its binding with a yet unidentified site which is present in the molecules of all AAbs (independently on their specificity) and absent in other human Igs.

In planta production of ELPylated spidroin-based proteins results in non-cytotoxic biopolymers.

BACKGROUND: Spider silk is a tear-resistant and elastic biopolymer that has outstanding mechanical properties. Additionally, exiguous immunogenicity is anticipated for spider silks. Therefore, spider silk represents a potential ideal biomaterial for medical applications. All known spider silk proteins, so-called spidroins, reveal a composite nature of silk-specific units, allowing the recombinant production of individual and combined segments. RESULTS: In this report, a miniaturized spidroin gene, named VSO1 that contains repetitive motifs of MaSp1 has been synthesized and combined to form multimers of distinct lengths, which were heterologously expressed as elastin-like peptide (ELP) fusion proteins in tobacco. The elastic penetration moduli of layered proteins were analyzed for different spidroin-based biopolymers. Moreover, we present the first immunological analysis of synthetic spidroin-based biopolymers. Characterization of the binding behavior of the sera after immunization by competitive ELISA suggested that the humoral immune response is mainly directed against the fusion partner ELP. In addition, cytocompatibility studies with murine embryonic fibroblasts indicated that recombinant spidroin-based biopolymers, in solution or as coated proteins, are well tolerated. CONCLUSION: The results show that spidroin-based biopolymers can induce humoral immune responses that are dependent on the fusion partner and the overall protein structure. Furthermore, cytocompatibility assays gave no indication of spidroin-derived cytotoxicity, suggesting that recombinant produced biopolymers composed of spider silk-like repetitive elements are suitable for biomedical applications.

Structure of an HIV-1-neutralizing antibody target, the lipid-bound gp41 envelope membrane proximal region trimer.

The membrane proximal external region (MPER) of HIV-1 glycoprotein (gp) 41 is involved in viral-host cell membrane fusion. It contains short amino acid sequences that are binding sites for the HIV-1 broadly neutralizing antibodies 2F5, 4E10, and 10E8, making these binding sites important targets for HIV-1 vaccine development. We report a high-resolution structure of a designed MPER trimer assembled on a detergent micelle. The NMR solution structure of this trimeric domain, designated gp41-M-MAT, shows that the three MPER peptides each adopt symmetric α-helical conformations exposing the amino acid side chains of the antibody binding sites. The helices are closely associated at their N termini, bend between the 2F5 and 4E10 epitopes, and gradually separate toward the C termini, where they associate with the membrane. The mAbs 2F5 and 4E10 bind gp41-M-MAT with nanomolar affinities, consistent with the substantial exposure of their respective epitopes in the trimer structure. The traditional structure determination of gp41-M-MAT using the Xplor-NIH protocol was validated by independently determining the structure using the DISCO sparse-data protocol, which exploits geometric arrangement algorithms that guarantee to compute all structures and assignments that satisfy the data.

Differential accessibility of a rotavirus VP6 epitope in trimers comprising type I, II, or III channels as revealed by binding of a human rotavirus VP6-specific antibody.

Previous human antibody studies have shown that the human VH1-46 antibody variable gene segment encodes much of the naturally occurring human B cell response to rotavirus and is directed to virus protein 6 (VP6). It is currently unknown why some of the VH1-46-encoded human VP6 monoclonal antibodies inhibit viral transcription while others do not. In part, there are affinity differences between antibodies that likely affect inhibitory activity, but we also hypothesize that there are differing modes of binding to VP6 that affect the ability to block the transcriptional pore on double-layered particles. Here, we used a hybrid method approach for antibody epitope mapping, including single-particle cryo-electron microscopy (cryo-EM) and enhanced amide hydrogen-deuterium exchange mass spectrometry (DXMS) to determine the location and mode of binding of a VH1-46-encoded antibody, RV6-25. The structure of the RV6-25 antibody-double-layered particle (DLP) complex indicated a very complex binding pattern that revealed subtle differences in accessibility of the VP6 epitope depending on its position in the type I, II, or III channels. These subtle variations in the presentation or accessibility of the RV VP6 capsid layer led to position-specific differences in occupancy for binding of the RV6-25 antibody. The studies also showed that the location of binding of the noninhibitory antibody RV6-25 on the apical surface of RV VP6 head domain does not obstruct the transcription pore upon antibody binding, in contrast to binding of an inhibitory antibody, RV6-26, deeper in the transcriptional pore.

[Glycopolymers of microorganisms: achievements and future research (review)].

This review presents the current literature data on the structure of peptidoglycans, lipopolysaccharides, teichoic acids, the mechanism of biological action of lipopolysaccharides, and the possibility of uising oligosaccharides for creation of glycoconjugate vaccines, as well as promising areas for further research of glycopolymers of microorganisms.

Gamma-delta T-cell responses during subcutaneous Mycobacterium avium subspecies paratuberculosis challenge in sensitized or naive calves using matrix biopolymers.

We have developed a model to explore the early immune response against Mycobacterium avium subspecies paratuberculosis (Map) infection in the bovine calf using subcutaneously placed liquid gel matrix biopolymer (matrigel) containing live Map. Matrigel rapidly polymerizes in vivo, retains recruited cellular infiltrates and soluble immune mediators, and can be rapidly removed 48 hours later and depolymerized for analysis. In this study, we examined early host immune events at matrigel/Map sites; recruited cells were evaluated by histopathology and flow cytometry, and cytokines were measured by flow cytometry, enzyme-linked immunosorbent assay, and Luminex bead immunoassay. Our results demonstrate earlier recruitment of gamma-delta (γδ) T cells to matrigel/Map challenge sites compared to CD4+ T cells. We also show that significantly more γδ T cells were recruited to matrigel/Map sites postinfection day 7 compared to postinfection day 30 and that these cells produced significant amounts of the cytokine interferon gamma. We also provide evidence that peripheral blood-derived γδ T-cell subsets in cattle differentially generate interferon gamma, suggesting distinct roles for these cells. These data provide unique insight into initial antimycobacterial host cellular immune responses following Map infection in calves.

Análisis de las polihidroxialcanoato sintasas (PhaC1 y PhaC2) en una cepa de Pseudomonas fluorescens IBUN S1602, aislada en suelos colombianos / Analysis of polyhydroxyalkanoate synthases (PhaC1 and PhaC2) in a strain of Pseudomonas fluorescens IBUN S1602 isolated from Colombian soil

La cepa Pseudomonas fluorescens IBUN S1602 conforma el grupo de aislamientos provenientes de suelos colombianos de caña de azúcar, que acumula polihidrioxialcanoato (PHA), fue seleccionada como promisoria para escalamiento comercial por tener afinidad por sustratos alternativos y económicos como el glicerol, aceites usados, suero de leche, entre otros. Dada la importancia de la enzima sintasa en la síntesis de los PHAs, en el presente trabajo se realizó el análisis molecular de los genes phaC1 y phaC2 que codifican las enzimas sintasas tipo II (PhaC1 y PhaC2). Para la obtención de los amplímeros requeridos en la secuenciación, se utilizó la técnica de PCR bajo condiciones estandarizadas para iniciadores diseñados reportados en las bases de datos. Se identificaron dos fragmentos de 1680 pb y 1683 pb correspondientes a phaC1 y phaC2. El análisis comparativo de las secuencias proteicas resultantes de estos genes demuestra que la sintasa IBUN S1602 contiene la región α/β hidrolasa y 8 residuos de aminoácidos conservados, que son características de las sintasas examinadas a nivel mundial. Se analizó la estructura enzimática a nivel primario y se predijo la secundaria. Se concluyó que las sintasas de la cepa Pseudomonas fluorescens IBUN S1602 presentan alta homología con las sintasas tipo II que se reportan para Pseudomonas. Los resultados obtenidos contribuyen al entendimiento básico de la biosíntesis de PHA, la cual permitirá, en un futuro, el aumento de la calidad de PHA debida a la modulación del nivel de sintasa que se exprese en un organismo recombinante, con el fin de variar el peso molecular del biopolímero, propiedad esencial en el estudio de aplicaciones industriales.

The strain Pseudomonas fluorescens IBUN S1602 forms the group of isolates from colombian sugarcane soil´s, which accumulates polyhydroxyalkanoate biopolymer (PHA) and was selected as promising for commercial scale by having affinity for economic and alternative substrates such as glycerol, oils, whey, among others. Given the importance of the synthase enzyme in the synthesis of PHAs, was realized the molecular analysis of genes phaC1 and phaC2 which encode type II synthases (PhaC1 y PhaC2). To obtain the amplimers required in the sequencing, was used the PCR technique under standardized conditions for primers designed based on the updated review in databases. Were identified two fragments of 1680 bp and 1683 bp for phaC1 and phaC2. Comparative analysis of the resulting protein sequences of these genes shows that the IBUN S1602 synthases containing the region α/β hydrolase and 8 conserved amino acid residues that are characteristic of synthases examined worldwide. Enzyme structure was analyzed at the primary level and was predicted the secondary. It is concluded that synthase strain Pseudomonas fluorescens IBUN S1602 has high homology with type II synthases that are reported for Pseudomonas. The results contribute to basic understanding of the biosynthesis of PHA, and will allow in the future, increasing the quality of PHA due to modulation of the level of synthase is expressed in a recombinant organism, in order to vary the weight molecular biopolymer, an essential property in the study of industrial applications.

Immune safety evaluation of polymerized porcine hemoglobin (pPolyHb): a potential red blood cell substitute.

Polymerized Porcine Hemoglobin (pPolyHb), a hemoglobin-based oxygen carrier (HBOC), was developed as a potential red blood substitute for clinical applications. Assessment of its effects on the immune system is an important component of the overall safety evaluation of HBOC. For this purpose, we assessed three inflammation indicators, including complement C3a, IL-6, and TNF-? in cultured cells and in a rat model when pPolyHb was incubated or administrated with the cells/animals. Our results suggested that the levels of these three indicators were not statistically changed upon pPolyHb stimulation, indicating that pPolyHb is not immunotoxic to cells and animals in this aspect.

Co-expression of two distinct polysialic acids, α2,8- and α2,9-linked polymers of N-acetylneuraminic acid, in distinct glycoproteins and glycolipids in sea urchin sperm.

Naturally occurring polysialic acid (polySia) structures have a large diversity, primarily arising from the diversity in the sialic acid components as well as in the intersialyl linkages. In 2004, we demonstrated the presence of a new type of polySia, 8-O-sulfated N-acetylneuraminic acid (Neu5Ac) capped α2,9-linked polyNeu5Ac, on the O-glycans of a major 40-80 kDa sialoglycoprotein, flagellasialin, in sea urchin sperm. In this study, we demonstrated that another type of polySia, the α2,8-linked polyNeu5Ac, exclusively occurs on O-glycans of a 190 kDa glycoprotein (190 kDa-gp), whereas the α2,9-linked polyNeu5Ac is exclusively present on flagellasialin. The 190 kDa-gp is localized in both flagellum and head of sperm. We also demonstrated that polysialogangliosides containing the α2,8-linked polyNeu5Ac are present in sperm head. Thus, this study shows two novel features of the occurrence of polySia in nature, the co-localization of polySia with different intersialyl linkages, the α2,8- and α2,9-linkages, in a single cell and the occurrence of α2,8-linked polyNeu5Ac in glycolipids. Anti-α2,8-linked polyNeu5Ac antibody had no effect on fertilization, which contrasted with the previous results that anti-α2,9-linked polyNeu5Ac antibody inhibited sperm motility and fertilization. Based on these properties, distinct functions of α2,8- and α2,9-polySia structures are implicated in fertilization.

Polymeric immunoglobulin receptor.

The surface of mucosal sites, such as the intestinal tract, are covered by epithelial cells. To protect the intestinal environment from invading pathogens and maintain homeostasis, the human body developed an exquisite acquired immune system, referred to as the mucosal immune system, in which epithelial cells and lymphocytes function cooperatively. The main player in this immune system is the polymeric immunoglobulins (pIgs), in particular dimeric IgA (dIgA). To exert its protective effect, dIgA produced in the lamina propria must be transported to the intestinal lumen across epithelial cells. This process is called transcytosis and is mediated by polymeric immunoglobulin receptor (pIgR), which is exclusively produced by intestinal epithelial cells (IECs). DIgA is captured by pIgR on the basolateral surface of IECs and transcytosed to the opposite side of IECs. The dIgA-pIgR complex is expressed on the apical surface of IECs and proteolytically cleaved to generate secretory IgA (SIgA). This review describes the current understanding and recent progress in this research field.

Immunomodulatory effects of Aureobasidium pullulans SM-2001 exopolymers on the cyclophosphamide-treated mice.

The immunomodulatory effects of exopolymers of Aureobasidium pullulans SM-2001 containing beta-1,3/1,6-glucan were evaluated on the cyclophosphamide (CPA)-treated mice. To induce immunosuppress, 150 and 110 mg/kg of CPA were intraperitoneally injected at 1 and 3 days before start of test material administrations, respectively. Exopolymers were subcutaneously or orally administered in a volume of 10 ml/kg, 4 times; 12-hr intervals from 24 hrs after second treatment of CPA. After treatment of exopolymers, the changes of thymus and spleen weights, splenic amounts of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and IL-10, thymic and splenic CD3+, CD4+, CD8+ and TNF-alpha+ cells were monitored in CPA-treated mice. As results of CPA treatment, dramatical decreases of the CD3+, CD4+, CD8+ and TNF-alpha+ cells were detected in thymus and spleen with decreases of thymus and spleen weights. In addition, decreases of splenic TNF-alpha, IL-1beta and IL-10 contents were also detected at flow cytometrical observations. However, oral and subcutaneous treatment of exopolymers effectively reduced the immunosuppressive changes induced by CPA. Therefore, it is concluded that exopolymers of A. pullulans can be effectively prevent the immunosuppress mediated, at least partially, recruitment of T cells and TNF-alpha+ cells or enhancement of their activity, and can provide effective prevention or treat regimes for the immunosuppress and related diseases such as cancer, sepsis and high-dose chemotherapy or radiotherapy.

Histological study on side effects and tumor targeting of a block copolymer micelle on rats.

Histological examinations were performed with polymeric micelle-injected rats for evaluations of possible toxicities of polymeric micelle carriers. Weight of major organs as well as body weight of rats was measured after multiple intravenous injections of polymeric micelles forming from poly(ethylene glycol)-b-poly(aspartate) block copolymer. No pathological toxic side effects were observed at two different doses, followed only by activation of the mononuclear phagocyte system (MPS) in the spleen, liver, lung, bone marrow, and lymph node. This finding confirms the absence of--or the very low level of--in vivo toxicity of the polymeric micelle carriers that were reported in previous animal experiments and clinical results. Then, immunohistochemical analyses with a biotinylated polymeric micelle confirmed specific accumulation of the micelle in the MPS. The immunohistochemical analyses also revealed, first, very rapid and specific accumulation of the micelle in the vasculatures of tumor capsule of rat ascites hepatoma AH109A, and second, the micelle's scanty infiltration into tumor parenchyma. This finding suggests a unique tumor-accumulation mechanism that is very different from simple EPR effect-based tumor targeting.

An antigenic epitope of influenza virus nucleoprotein (NP) associated with polymeric forms of NP.

Intracellular influenza virus nucleoprotein (NP) is characterized by a high efficiency of homo-polymers formation, however their antigenic structure is still incompletely known. Herein, we report that RNase-resistant intracellular NP homo-polymers have a highly ordered conformational antigenic epitope, which depends on inter-subunit interactions of monomeric NPs. Our studies have shown that in radioimmunoprecipitation (RIPA) intracellular NP polymers bind mAb N5D3 and RNase does not prevent their mAb binding. In contrast to NP polymers, NP monomeric subunits, obtained by thermo-dissociation of NP polymers, fail to bind the mAb N5D3 in RIPA. At the same time, the in vitro concentration of thermo-denatured monomeric NPs in both soluble and immobilized forms results in NP-NP association, accompanied by renaturation of the N5D3 epitope. The same results were detected by Western blotting, where the pre-denatured NP monomers were concentrated on nitrocellulose into a single 56 kDa band, which then caused NP-NP self-association as well as N5D3 epitope renaturation. Thus, the in vitro renaturation of N5D3 epitope is markedly dependent on NP monomers concentration. The results obtained suggest that in vivo formation and in vitro renaturation of the N5D3 epitope depend on inter-subunit interactions of monomeric NPs and NP-NP interactions influence the antigenic structure of the influenza virus NP polymers.

Anti-oligomeric single chain variable domain antibody differentially affects huntingtin and alpha-synuclein aggregates.

Huntington's and Parkinson's diseases are both neurodegenerative disorders caused at least in part by misfolding and aggregation of huntingtin (htt) and alpha-synuclein, respectively. Here we use a single chain antibody fragment (scFv) isolated against oligomeric alpha-synuclein to probe similarities and differences between the aggregation and toxic mechanisms of htt and alpha-synuclein. When incubated with htt, the scFv both blocks formation of and promotes dissociation of fibrillar aggregates, but stabilizes formation of cytotoxic oligomeric aggregates. Previous studies with monomeric alpha-synuclein showed the scFv prevented fibrillar aggregation, but blocked toxicity of oligomeric aggregates. These divergent effects suggest the toxic mechanisms of oligomeric aggregates differ among amyloidogenic protein species.

[Immunogenicity of surface and capsular antigens of Burkholderia].

Study showed that five (C3, C6, C9, C10, C11) out of ten chromatographic fractions of surface and capsular antigens of B. mallei significantly stimulated cell-mediated immunity that manifested in activation of delayed hypersensivity reactions (DHS) and phagocyteability of noncapsulated avirulent strain of B. mallei with added surface and capsular antigenic complexes. Other fractions did not stimulate cell-mediated immunity, furthermore, fraction C8, which contained capsular biopolymer with mass of 200 kD (Ar8), was characterized by immunosuppressive effect on DHS and phagocytosis. Observed stimulation of cell-mediated immunity by fractions referred above has been confirmed by assessment of their protective effects on the model of experimental melioidosis in white rats. Relationship between markers of humoral and cell-mediated immunity, including markers of specific response, was not observed.

Chemical characteristics and immuno-modulating activities of exo-biopolymers produced by Grifola frondosa during submerged fermentation process.

The immuno-modulating activities and chemical characteristics of exo-biopolymer (EX-GF) produced by a submerged mycelial culture of Grifola frondosa were studied. The EX-GF was fractionated into EX-GF-Fr.I, II, and III by Sephadex G-100 gel chromatography. Anti-complementary activity of EX-GF-Fr.III was highest (71.1%) among them, and its activation system occurred through both classical and alternative pathways, where the classical pathway found to be major one. Lysosomal enzyme activity and nitric oxide production ability of macrophage were also found to be mediated by EX-GF-Fr.III. The molecular weight of the EX-GF-Fr.I, II, and III was estimated to be about 163, 40, and 2.8 kDa, respectively. Total sugar and protein contents of the three fractions were 80.3, 61.9 and 89.3%, and 17.3, 35.2, and 10.7%, respectively. The sugar and amino acid compositions of the EX-GF-Fr.I, II, and III were also analyzed in detail.

Chemical characteristics and immuno-stimulating properties of biopolymers extracted from Acanthopanax sessiliflorus.

During our search for macrophage stimulating compounds from medicinal plants, we isolated biopolymers from Acanthopanax sessiliflorus. Isolated fraction AS-5 showed maximum potential, and stimulated lysosonal enzymatic activity by 230% at 300 microg/ml. The nitric oxide (NO) producing ability of AS-5 100 microg/ml was 58 microM when treated with interferon-gamma and lipopolysaccharide 20 micro/ml. The lymphocyte proliferating effects of isolated biopolymer fractions were also investigated. Highest lymphoproliferative activity (a 2.8-fold enhancement compared to salines treated group was exhibited by AS-3 at 200 micro/ml followed by AS-5 and AS-6. The AS-3 fraction stimulated only T-lymphocytes and had little or no effect on B-lymphocyte proliferation. Partially methylated alditol acetates were prepared to elucidate the glycosyl linkage-compositions of the AS-3 and AS-5 biopolymers, and were analyzed by GC-MS. The AS-3 and AS-5 biopolymer fractions were found to contain 2,3,4-tri-O-methyl-D-glucitol, 2,3,4-tri-O-methyl-D-galacitol 3,4,6-tri-O-methyl-galacitol, 2-O-methyl-arabinitol and 2,4,6-tri-O-methyl-D-glucitol, 2,3,6-tri-O-methyl-D-galacitol linkages, respectively.

A polymeric bacterial protein activates dendritic cells via TLR4.

The enzyme lumazine synthase from Brucella spp. (BLS) is a highly immunogenic protein that folds as a stable dimer of pentamers. It is possible to insert foreign peptides and proteins at the 10 N terminus of BLS without disrupting its general folding, and these chimeras are very efficient to elicit systemic and oral immunity without adjuvants. In this study, we show that BLS stimulates bone marrow dendritic cells from mice in vitro to up-regulate the levels of costimulatory molecules (CD40, CD80, and CD86) and major histocompatibility class II Ag. Furthermore, the mRNA levels of several chemokines are increased, and proinflammatory cytokine secretion is induced upon exposure to BLS. In vivo, BLS increases the number of dendritic cells and their expression of CD62L in the draining lymph node. All of the observed effects are dependent on TLR4, and clearly independent of LPS contamination. The described characteristics of BLS make this protein an excellent candidate for vaccine development.