Sugar-Modified Poly(propylene imine) Dendrimers Stimulate the NF-κB Pathway in a Myeloid Cell Line.

PURPOSE: Fourth-generation poly(propylene imine) dendrimers fully surface-modified by maltose (dense shell, PPI-m DS) were shown to be biocompatible in cellular models, which is important for their application in drug delivery. We decided to verify also their inherent bioactivity, including immunomodulatory activity, for potential clinical applications. We tested their effects on the THP-1 monocytic cell line model of innate immunity effectors. METHODS: To estimate the cytotoxicity of dendrimers the reasazurin assay was performed. The expression level of NF-κB targets: IGFBP3, TNFAIP3 and TNF was determined by quantitative real-time RT-PCR. Measurement of NF-κB p65 translocation from cytoplasm to nucleus was conducted with a high-content screening platform and binding of NF-κB to a consensus DNA probe was determined by electrophoretic mobility shift assay. The cytokine assay was performed to measure protein concentration of TNFalpha and IL-4. RESULTS: We found that PPI-m DS did not impact THP-1 viability and growth even at high concentrations (up to 100 µM). They also did not induce expression of genes for important signaling pathways: Jak/STAT, Keap1/Nrf2 and ER stress. However, high concentrations of 4th generation PPI-m DS (25-100 µM), but not their 3rd generation counterparts, induced nuclear translocation of p65 NF-κB protein and its DNA-binding activity, leading to NF-κB-dependent increased expression of mRNA for NF-κB targets: IGFBP3, TNFAIP3 and TNF. However, no increase in pro-inflammatory cytokine secretion was detected. CONCLUSION: We conclude that maltose-modified PPI dendrimers of specific size could exert a modest immunomodulatory effect, which may be advantageous in clinical applications (e.g. adjuvant effect in anti-cancer vaccines).

Maltooligosaccharides from JEG-3 trophoblast-like cells exhibit immunoregulatory properties.

PROBLEM: to better understand the immunoregulatory properties of trophoblasts, we have searched for small immunologically active carbohydrates derived from intact trophoblast-like cells. METHOD OF STUDY: using solid phase extraction coupled with HPLC and mass spectrometry methods, we have characterized a low molecular weight carbohydrate-rich fraction associated with JEG-3 cells. We have also tested the bioactivities of selected authentic oligosaccharides found in the oligosaccharide fraction. RESULTS: the most abundant components of the low molecular weight carbohydrate-rich fraction were maltotriose and maltotetraose, with detectable amounts of maltopentaose. When authentic maltooligosaccharides were tested using lymphocytes, IL-2 inhibition was observed. This activity was dependent upon the number of saccharide subunits, stereochemistry, and concentration. To further test maltooligosaccharide properties, maltopentose was attached to glass cover slips. Although spontaneous neutrophil motility was observed on unmodified and control surfaces, it was inhibited on maltooligosaccharide-derivatized surfaces. CONCLUSION: maltooligosaccharides are associated with the trophoblast's surface where they may exhibit immunoregulatory activities.

Naked plasmid DNA formulation: effect of different disaccharides on stability after lyophilisation.

Since plasmid DNA (pDNA) is unstable in solution, lyophilisation can be used to increase product shelf life. To prevent stress on pDNA molecules during lyophilisation, cryo- and lyoprotectants have to be added to the formulation. This study assessed the effect of disaccharides on naked pDNA stability after lyophilisation using accelerated stability studies. Naked pDNA was lyophilised with sucrose, trehalose, maltose or lactose in an excipient/DNA w/w ratio of 20. To one part of the vials extra residual moisture was introduced by placing the vials half opened in a 25 degrees C/60% RH climate chamber, before placing all vials in climate chambers (25 degrees C/60% RH and 40 degrees C/75% RH) for stability studies. An ex vivo human skin model was used to assess the effect of disaccharides on transfection efficiency. Lyophilisation resulted in amorphous cakes for all disaccharides with a residual water content of 0.8% w/w. Storage at 40 degrees C/75% RH resulted in decreasing supercoiled (SC) purity levels (sucrose and trehalose maintained approximately 80% SC purity), but not in physical collapse. The addition of residual moisture (values between 7.5% and 10% w/w) resulted in rapid collapse except for trehalose and decreasing SC purity for all formulations. In a separate experiment disaccharide formulation solutions show a slight but significant reduction (<3% with sucrose and maltose) in transfection efficiency when compared to pDNA dissolved in water. We demonstrate that disaccharides, like sucrose and trehalose, are effective lyoprotectants for naked pDNA.

Exploitation of a monoclonal antibody for weak affinity-based separation in capillary gel electrophoresis.

Weak biospecific recognition has been established for affinity separation in high performance liquid chromatography (HPLC). The use of weak affinity chromatography (WAC) has been limited previously by the insufficient separation efficiency achieved, allowing only some 1000 plates/m to be obtained. However, it has been shown that chiral drug separation can be performed with capillary affinity gel electrophoresis (CAGE) at considerably improved efficiency as compared with traditional chromatographic procedures. Our present study demonstrates the potential of weak affinity monoclonal antibodies as a generic method for immunologically based separations in capillary electrophoresis. Monoclonal antibodies were polymerized within a silica capillary and were used for the separation of structurally similar carbohydrate antigens. The results indicate that weak biospecific interactions can be utilized in a CAGE format to produce highly selective separation of the alpha- and beta-forms of p-nitrophenyl-labeled maltose. It remains to be seen, however, how efficient weak affinity separation in CAGE can be compared with affinity HPLC protocols. Details of typical separations and of the preparation of the antibody gel are presented.

Anaphylactoid reaction to maltose 5% solution during spinal anaesthesia.

PURPOSE: A rare case of an anaphylactoid reaction to maltose solution is presented. CLINICAL FEATURES: A 28-yr-old man underwent repair of bilateral inguinal hernia under spinal anaesthesia with dibucaine. At the end of operation, he developed generalized flush and circulatory collapse immediately after receiving Na acetate solution containing maltose, 5%, i.v. The reactions were treated with 32 mg ephedrine and 250 mg methylprednisolone i.v., and rapid infusion of 1,000 ml acetated Ringer's solution. The skin tests provoked positive responses to maltose solutions. CONCLUSION: The clinical features and skin tests suggested that the episode was an anaphylactoid reaction to maltose. Maltose is one of the dissacharides (MW: 342) produced from starch and glycogen. Maltose solutions are used frequently in Japan as a carbohydrate source. Further study is required to confirm whether maltose has an immunological antigen-eliciting activity.

Modulating the immunological properties of a linear B-cell epitope by insertion into permissive sites of the MalE protein.

In a previous study, a set of positions in the MalE protein from Escherichia coli were identified, which tolerated short insertions or deletions without compromising the maltose binding activity of the protein. It is now shown that these sites accommodate an insert of 13 amino acids and are, therefore, permissive. Eleven sites were used, including eight permissive sites, to display a linear neutralization B-cell epitope of poliovirus (C3 epitope) at different positions on the surface of MalE. The affinity of a monoclonal neutralizing anti-poliovirus antibody (anti-C3 mAb) for the hybrid proteins varied from undetectable, to more than 1000 times higher than for the synthetic peptide. Therefore, some MalEC3 proteins mimic interactions of the viral epitope with the monoclonal antibody more efficiently than the free peptide. The results are interpreted in terms of the mobility of the insert and its flanking regions. It was further shown that some of the purified hybrid proteins are able to induce high titer anti-C3-peptide antibodies in mice. A strong correlation exists between the capacity of a MalEC3 protein to induce anti-C3-peptide antibodies and the antigenicity of the inserted peptide, measured with a polyclonal serum raised against the synthetic peptide.

Interaction of anti-kojibiose antibody with the lipoteichoic acids from Streptococcus faecalis and Streptococcus faecium.

Antisera prepared in rabbits by immunization with p-aminophenyl beta-kojibioside conjugated to bovine serum albumin (antikojibiose sera), readily agglutinated whole cells of Streptococcus faecalis or Streptococcus faecium, and showed specific reactions with the lipoteichoic acids (LTAs) of these streptococci by passive hemagglutination, microscale enzyme-linked immunosorbent assay, and crossed immunoelectrophoresis. The interaction of the antikojibiose sera with the LTAs was inhibited best by kojibiose [alpha-D-glucopyranosyl-(1----2)-D-glucose], somewhat less by the dextran from which the kojibiose was prepared, and not measurably by maltose [alpha-D-glucopyranosyl-(1----4)-D-glucose]. The sera reacted only minimally in only the most sensitive assay (microscale enzyme-linked immunosorbent assay) with LTA from group A streptococci (this LTA contains a single kojibiosyl residue as part of the glycolipid moiety of the molecule and failed to react with the Lactobacillus fermentum LTA which is substituted with alpha-D-galactopyranosyl-(1----2)-D -glucosyl units.