Nanoparticulate Tubular Immunostimulating Complexes: Novel Formulation of Effective Adjuvants and Antigen Delivery Systems.

New generation vaccines, based on isolated antigens, are safer than traditional ones, comprising the whole pathogen. However, major part of purified antigens has weak immunogenicity. Therefore, elaboration of new adjuvants, more effective and safe, is an urgent problem of vaccinology. Tubular immunostimulating complexes (TI-complexes) are a new type of nanoparticulate antigen delivery systems with adjuvant activity. TI-complexes consist of cholesterol and compounds isolated from marine hydrobionts: cucumarioside A2-2 (CDA) from Cucumaria japonica and monogalactosyldiacylglycerol (MGDG) from marine algae or seagrass. These components were selected due to immunomodulatory and other biological activities. Glycolipid MGDG from marine macrophytes comprises a high level of polyunsaturated fatty acids (PUFAs), which demonstrate immunomodulatory properties. CDA is a well-characterized individual compound capable of forming stable complex with cholesterol. Such complexes do not possess hemolytic activity. Ultralow doses of cucumariosides stimulate cell as well as humoral immunity. Therefore, TI-complexes comprising biologically active components turned out to be more effective than the strongest adjuvants: immunostimulating complexes (ISCOMs) and complete Freund's adjuvant. In the present review, we discuss results published in series of our articles on elaboration, qualitative and quantitative composition, ultrastructure, and immunostimulating activity of TI-complexes. The review allows immersion in the history of creating TI-complexes.

[Enhancing the immunogenic activity of influvac vaccine in the use of adjuvant TI complexes modified by echinochrome A].

The self-assembly of marine macrophyte glycolipids, holothurian saponin, and cholesterol gave rise to nanoscale morphological structures called tubular immunostimulating (TI) complexes. Whether the latter could be used on the basis of vaccine preparations containing the influenza virus subunit antigens was studied. There was an obvious increase in the immunogenicity of influenza virus hemagglutinin when the experimental animals were immunized with this antigen as part of TI complexes. It was shown that the adjuvant activity of the TI complex to influenza virus hemagglutinin could be enhanced by adding the known antioxidant echinochrome A from a sand-dollar (Echinarachnius parma) to the matrix of the TI complex.

Tubular immunostimulating complex based on cucumarioside A2-2 and monogalactosyldiacylglycerol from marine macrophytes.

BACKGROUND: There is an urgent need to develop safe and effective adjuvants for the new generation of subunit vaccines. We developed the tubular immunostimulating complex (TI-complex) as a new nanoparticulate antigen delivery system. The morphology and composition of TI-complexes principally differ from the known vesicular immunostimulating complexes (ISCOMs). However, methodology for the preparation of TI-complexes has suffered a number of shortcomings. The aim of the present work was to obtain an antigen carrier consisting of triterpene glycosides from Cucumaria japonica, cholesterol, and monogalactosyldiacylglycerol from marine macrophytes with reproducible properties and high adjuvant activity. RESULTS: The cucumarioside A2-2 - cholesterol - MGalDG ratio of 6:2:4 (by weight) was found to provide the most effective formation of TI-complexes and the minimum hemolytic activity in vitro. Tubules of TI-complexes have an outer diameter of about 16 nm, an inner diameter of 6 nm, and a length of 500 nm. A significant dilution by the buffer gradually destroyed the tubular nanoparticles. The TI-complex was able to increase the immunogenicity of the protein antigens from Yersinia pseudotuberculosis by three to four times. CONCLUSIONS: We propose an optimized methodology for the preparation of homogeneous TI-complexes containing only tubular particles, which would achieve reproducible immunization results. We suggest that the elaborated TI-complexes apply as a universal delivery system for different subunit antigens within anti-infectious vaccines and enhance their economic efficacy and safety.

Subcellular localization and dynamics of a digalactolipid-like epitope in Toxoplasma gondii.

Toxoplasma gondii is a unicellular parasite characterized by unique extracellular and intracellular membrane compartments. The lipid composition of subcellular membranes has not been determined, limiting our understanding of lipid homeostasis, control, and trafficking, a series of processes involved in pathogenesis. In addition to a mitochondrion, Toxoplasma contains a plastid called the apicoplast. The occurrence of a plastid raised the question of the presence of chloroplast galactolipids. Using three independent rabbit and rat antibodies against digalactosyldiacylglycerol (DGDG) from plant chloroplasts, we detected a class of Toxoplasma lipids harboring a digalactolipid-like epitope (DGLE). Immunolabeling characterization supports the notion that the DGLE polar head is similar to that of DGDG. Mass spectrometry analyses indicated that dihexosyl lipids having various hydrophobic moieties (ceramide, diacylglycerol, and acylalkylglycerol) might react with anti-DGDG, but we cannot exclude the possibility that more complex dihexosyl-terminated lipids might also be immunolabeled. DGLE localization was analyzed by immunofluorescence and immunoelectron microscopy and confirmed by subcellular fractionation. No immunolabeling of the apicoplast could be observed. DGLE was scattered in pellicle membrane domains in extracellular tachyzoites and was relocalized to the anterior tip of the cell upon invasion in an actin-dependent manner, providing insights on a possible role in pathogenetic processes. DGLE was detected in other Apicomplexa (i.e., Neospora, Plasmodium, Babesia, and Cryptosporidium).

[Immunostimulatory characteristics of a novel carrier on the basis of cucumarioside A2-2 and monogalactosyldiacylgycerol].

A novel antigen carrier has been formulated on the basis of a cucumarioside-A2-2 triterpene glycoside (CD) complex with cholesterol and monogalactosyldiacylglycerol from Ahnfeltia tobuchiensis (MGDG(At)) and Ulva fenestrate (MGDG(Uf)). Morphological and immunostimulative characteristics of the carrier were studied. Electron microscopy experiments demonstrated the formation of homogeneous tubular structures in a mixture of CD, cholesterol, and MGDG in molar ratio of 1:2:3. In animals immunized by the carrier bearing pore forming protein monomer of pseudotuberculosis agent CD and MGDG synergically affected synthesis of specific antibodies, interleukin-2, and gamma-interferon and delayed hypersensitivity reaction when compared to Freund's complete adjuvant or to immunostimulatory complexes between Quillaja saponaria saponins and phosphatidylcholine from egg yolk. The immunostimulatory effect depends upon the composition of polyunsaturated fatty acids of MGDG. The new tubular adjuvant carrier is a competitive adjuvant, as it includes CD obtained from far-eastern sea cucumber commercial species Cucumaria japonica, and MGDG from seaweed.

A long trek down the pathways of cell death in Alzheimer's disease.

The invitation to contribute to the issue marking the 100th anniversary of Alzheimer's disease gave me pause to reflect on the significant milestones in my own research. This brief and personal description of my laboratory's search for the cause of cell dysfunction and death in Alzheimer's disease marks only highlights, and my apologies to those whose work I have passed over.

Acquisition of arylsulfatase A onto the mouse sperm surface during epididymal transit.

Arylsulfatase A (AS-A) is localized to the sperm surface and participates in sperm-zona pellucida binding. We investigated how AS-A, usually known as an acrosomal enzyme, trafficked to the sperm surface. Immunocytochemistry of the mouse testis confirmed the existence of AS-A in the acrosomal region of round and elongating spermatids. However, immunofluorescence and flow cytometry indicated the absence of AS-A on the surface of live testicular sperm. In contrast, positive AS-A staining was observed in the heads of live caudal epididymal and vas deferens sperm. The results suggested that acquisition of AS-A on the sperm surface occurred during epididymal transit. Immunocytochemistry of the epididymis revealed AS-A in narrow and apical cells in the initial segment and in clear cells in all epididymal regions. However, these epithelial cells are in the minority and are not involved in secretory activity. In the caudal epididymis and vas deferens, AS-A was also localized to principal cells, the major epithelial cells. Because principal cells have secretory activity, they may secrete AS-A into the epididymal fluid. This hypothesis was supported by our results revealing the presence of AS-A in the epididymal and vas deferens fluid (determined by immunoblotting and ELISA) and an AS-A transcript in the epididymis (by reverse transcription polymerase chain reaction). Alexa-430 AS-A bound to epididymal sperm with high affinity (Kd = 46 nM). This binding was inhibited by treatment of sperm with an antibody against sperm surface sulfogalactosylglycerolipid. This finding suggests that AS-A in the epididymal fluid may deposit onto sperm via its affinity to sulfogalactosylglycerolipid.

Binding of arylsulfatase A to mouse sperm inhibits gamete interaction and induces the acrosome reaction.

We have shown previously that male germ cell-specific sulfoglycolipid, sulfogalactosylglycerolipid (SGG), is involved in sperm-zona pellucida binding, and that SGG and its desulfating enzyme, arylsulfatase A (AS-A), coexist in the same sperm head area. However, AS-A exists at a markedly low level in sperm as compared to SGG (i.e., 1/400 of SGG molar concentration). In the present study, we investigated whether perturbation of this molar ratio would interfere with sperm-egg interaction. We demonstrated that purified AS-A bound to the mouse sperm surface through its high affinity with SGG. When capacitated, Percoll gradient-centrifuged mouse sperm were treated for 1 h with various concentrations of AS-A, their binding to zona-intact eggs was inhibited in a dose-dependent manner and reached the background level with 63 nM AS-A. This inhibition could be partially explained by an increase in premature acrosome reaction. The acrosome-reacted sperm population of the 63 nM AS-A-treated sperm sample was twice that of the control sample (treated with 63 nM ovalbumin) at 1 h (i.e., 32% vs. 15%) and rose to 53% at 2 h. This induction was presumably due to SGG aggregation attributed to AS-A, existing as a dimer at neutral pH, and could be mimicked by anti-SGG immunoglobulin (Ig) G/IgM + secondary IgG antibody. Drastic inhibition (75%) of in vivo fertilization was also observed in females inseminated with sperm suspension containing 630 nM AS-A as compared to the rate in females inseminated with sperm suspension included with 630 nM ovalbumin. Our results demonstrate a promising potential for AS-A as a nonhormonal, vaginal contraceptive.

[In vitro hepatic targeting tendency of galactosyl-anti CD3 McAb-TILS].

This study was undertaken to enhance the hepatic targeting tendency of tumor infiltrating lymphocytes (TILs) and hence to lower the recurrence rate of primary liver cancer after hepatectomy. Galactosyl-anti CD3McAb-TILs were prepared and then were incubated together with hepatocytes. Their interaction through asialoglycoprotein receptor-mediated mechanism was observed under the inverted phase contrast microscope. The carbohydrate density of galactosyl-anti CD3McAb and the combining rate of galactosyl-anti CD3McAb with TILs were measured. The results revealed that galactosyl-anti CD3 McAb-TIL obviously were adhered to hepatocytes. The carbohydrate density of galactosyl-anti CD3McAb was 62.18, and the combining rate of galactosyl-anti CD3McAb with TILs was 97.9%. The results suggested that in vitro hepatic targeting tendency of galactosyl-anti CD3McAb-TILs was satisfactory, and that the carbohydrate density of 62.18 and the combining rate of 97.9% ensured the effective use of TILs.