Photodynamic activity of anthraquinones isolated from Heterophyllaea pustulata Hook f. (Rubiaceae) on MCF-7c3 breast cancer cells.

Searching for agents that could be effective in the treatment of cancer, special highlight has focused on the study of numerous plant-derived compounds. We previously demonstrated that anthraquinones (AQs) isolated from a vegetal species: Heterophyllaea pustulata Hook f. (Rubiaceae), such as rubiadin, rubiadin-1-methyl ether, soranjidiol, soranjidiol-1-methyl ether exhibit photosensitizing properties without antecedents as photodynamic agents in malignant cells. In the present study, we investigated the potential role of these AQs as a phototoxic agent against human breast carcinoma using MCF-7c3 cells. All AQs exhibited significant photocytotoxicity on cancer cells at the concentration of 100 µM with 1 J/cm(2) light dose, resulting soranjidiol-1-methyl ether in complete cell destruction. The observed cellular killing by photoactivated AQs exhibited close relation with singlet oxygen production, except for soranjidiol-1-methyl ether, where cell viability decrease is in relation to uptake by tumor cells.

Determination of radionuclides in environmental test items at CPHR: traceability and uncertainty calculation.

Information about how the laboratory of Centro de Protección e Higiene de las Radiaciones (CPHR), Cuba establishes its traceability to the International System of Units for the measurement of radionuclides in environmental test items is presented. A comparison among different methodologies of uncertainty calculation, including an analysis of the feasibility of using the Kragten-spreadsheet approach, is shown. In the specific case of the gamma spectrometric assay, the influence of each parameter, and the identification of the major contributor, in the relative difference between the methods of uncertainty calculation (Kragten and partial derivative) is described. The reliability of the uncertainty calculation results reported by the commercial software Gamma 2000 from Silena is analyzed.

Cadaverine prevents the escape of Shigella flexneri from the phagolysosome: a connection between bacterial dissemination and neutrophil transepithelial signaling.

Shigella flexneri causes bacillary dysentery in humans by invading epithelial cells of the colon, which is characterized by an acute polymorphonuclear leukocyte (PMNL)-rich inflammation. Our recent studies demonstrated that cadaverine, a polyamine, specifically acts to abrogate transepithelial signaling to PMNL induced by S. flexneri. Here, insight is provided into the cellular mechanisms by which cadaverine attenuates the ability of Shigella species to induce PMNL signaling. It was found that cadaverine retards the lysis of the Shigella species-containing vacuole, suggesting that a blockade is established, in which the pathogen is prevented from adequately interacting with the cytoskeleton. Furthermore, an IcsA mutant of S. flexneri that cannot interact with the cytoskeleton and spreads intercellularly fails to induce transmigration of PMNL. Results indicate that cadaverine-induced compartmentalization of Shigella species to the phagolysosome might be a protective response of the host that directly contributes to the diminished ability of PMNL to transmigrate across model intestinal epithelia.

Homology of ab1 and ab3 monoclonal antibodies that neutralize Semliki Forest virus.

A noninternal image monoclonal antiidiotypic antibody (ab2 mAb), designated 1.13A321, that had proved its efficacy as vaccine against infection with Semliki Forest virus (SFV) in BALB/c mice, was used as immunogen to generate a panel of SFV-neutralizing monoclonal anti-anti-idiotypic antibodies (ab3 mAbs) to compare them genetically with ab1 mAb 1.13 (IgG2a). There are various studies that compare ab1 and ab3 mAbs but none that compare virus-neutralizing ab1 and ab3 mAbs. Five SFV-neutralizing ab3 MAbs, all IgG1, were obtained. The Vh gene (36-60), the D gene (Sp2), and the J gene (Jh2) encoding the heavy chain variable regions of all six mAbs, were similar and showed a high homology in the nucleotide sequence. The CDR3 amino acid sequences of four of five ab3 mAbs were identical to that of mAb1. One ab3 differed one amino acid in the CDR3 region. The results suggest that a strict selection criterion (virus neutralization) is sufficient to reach complete homology in the CDR3 region of mAb3. Future experiments are focused on selection of synthetic peptides in the CDR3 region as neutralizing mini-antibodies.

Induction of HIV-1 IIIb neutralizing antibodies in BALB/c mice by a chimaeric peptide consisting of a T-helper cell epitope of Semliki Forest virus and a B-cell epitope of HIV.

A colinearly synthesized peptide consisting of a H-2d restricted T-helper cell epitope of Semliki Forest virus (SFV) and triple repeats of sequence GPGRAF, derived from the V3 domain of HIV-1 strains, was used to immunize BALB/c (H-2d) mice. Pepscan analysis of sera from peptide-immunized mice revealed that the chimaeric peptide GREKFTIRPHYGKEIGPGRAFGPGRAFGPGRAF contains three distinct antibody-reactive sequences GREKFTIR, PHYGKEI and GPGRAF. The chimaeric peptide evoked HIV-1 IIIb neutralizing antibodies in serum as measured in vitro by reduction of syncytia formation and reduction of p24 production as well. So, the T-helper cell epitope of SFV provided help to a small linear neutralization epitope of HIV-1 strains. Interestingly, the T-helper cell epitope alone might induce antibodies cross-reactive with HIV-1 IIIb specific peptide GPGRAFVTIGK which shows some homology (residues underlined) with the antibody-reactive sequence GREKTIR of SFV.

Epitope polarity and adjuvants influence the fine specificity of the humoral response against Semliki Forest virus specific peptide vaccines.

The humoral response to synthetic peptide vaccines against Semliki Forest virus (SFV) in H-2d BALB/c mice was investigated with the enzyme linked immunosorbent assay and the pepscan technique. The peptide vaccines consisted of amino acid sequences 240-255 (B) and 137-151 (T) of the E2 membrane protein of SFV colinearly synthesized in either orientation T-B or B-T. Sequence B contains an epitope inducing humoral immunity to lethal SFV infection and sequence T contains a H-2d restricted T-helper cell epitope. With sera from mice immunized subcutaneously with peptide T-B, and Quil A as adjuvant, two immunodominant B-cell epitopes were identified, FVPRAD, at position 240-246 and PHYGKEI, at position 145-151. However, with peptide B-T and Quil A as adjuvant for immunization the epitope PHYGKEI was clearly immunodominant, but antibodies elicited against this epitope were not reactive with SFV-infected L cells in contrast to the antibodies elicited by epitope FVPRAD. An additional epitope EPARKGKVH, at position 247-255, was identified with sera from mice immunized subcutaneously with either peptide T-B or B-T and Montanide ISA 740 as an adjuvant. Monoclonal antibodies selected for reactivity with SFV-infected L cells did bind also to epitope FVPRAD. Interestingly, this epitope could induce antibodies cross-reactive with a synthetic peptide derived from macrophage migration inhibitory factor that shares amino acid residues VPRA at position 9-12 with the protective B-cell epitope FVPRAD. The present study shows clearly that the fine specificity of the humoral response against peptide vaccines is differentially influenced by both adjuvant and epitope polarity which may affect vaccine efficacy. Further, the study reminds us that potentially autoimmune antibodies could be induced by vaccines.

Epitope specificity of rabbit immunoglobulin G (IgG) elicited by pneumococcal type 23F synthetic oligosaccharide- and native polysaccharide-protein conjugate vaccines: comparison with human anti-polysaccharide 23F IgG.

Streptococcus pneumoniae type 23F capsular polysaccharide (PS23F) consitss of a repeating glycerol-phosphorylated branched tetrasaccharide. The immunogenicities of the following related antigens were investigated: (i) a synthetic trisaccharide comprising the backbone of one repeating unit, (ii) a synthetic tetrasaccharide comprising the complete repeating unit, and (iii) native PS23F (all three conjugated to keyhole limpet hemocyanin [KLH]) and (iv) formalin-killed S. pneumoniae 23F. All antigens except the trisaccharide-KLH conjugate induced relatively high anti-PS23F antibody levels in rabbits. The epitope specificity of such antibodies was then studied by means of an inhibition immunoassay. The alpha(1-->2)-linked L-rhamnose branch was shown to be immunodominant for immunoglobulin G (IgG) induced by tetrasaccharide-KLH, PS23F-KLH, and killed S. pneumoniae 23F: in most sera L-rhamnose totally inhibited the binding of IgG to PS23F. Thus, there appears to be no major difference in epitope specificity between IgG induced by tetrasaccharide-KLH and that induced by antigens containing the polymeric form of PS23F. Human anti-PS23F IgG (either vaccine induced or naturally acquired) had a different epitope specificity: none of the inhibitors used, including L-rhamnose and tetrasaccharide-KLH, exhibited substantial inhibition. These observations suggest that the epitope recognized by human IgG on PS23F is larger than the epitope recognized by rabbit IgG. Both human and rabbit antisera efficiently opsonized type 23F pneumococci, as measured in a phagocytosis assay using human polymorphonuclear leukocytes.

A shared idiotope among antibodies against Semliki Forest virus.

In the present study a shared idiotope was found among antibodies against a previously defined linear B-cell epitope of Semliki Forest virus (SFV). The synthetic B-cell epitope, located at amino acid positions 240 to 255 of the E2 membrane protein, was linked to an H-2d-restricted T-helper cell epitope of either SFV or influenza virus. Colinearly synthesized peptides of T-B polarity mixed with adjuvant were used to immunize BALB/c (H-2d) mice. After one booster immunization with either chimaeric peptide high serum antibody titers were measured against both synthetic peptide (240-255) and glutaraldehyde-fixed SFV-infected L cells. Against the synthetic peptide (240-255) a variety of monoclonal antibodies (MAbs) were produced that differed in reactivity with SFV, varied in heavy chain family, isotype, isoelectric point, and idiotype. Against one of the antipeptide MAbs (I02), that strongly reacted with SFV-infected L cells, an antiidiotypic MAb (ab2MAb), designated I02A3, was produced that could be inhibited in its binding to MAb I02 by the synthetic B-cell epitope. Therefore it was concluded that ab2 MAb I02A3 recognizes an idiotope closely associated with the antigen combining site of antipeptide MAb I02. This idiotope was definitively shared by two out of 15 antipeptide MAbs and by SFV-reactive antibodies present in both antipeptide sera and SFV-immune sera.

Influence of epitope polarity and adjuvants on the immunogenicity and efficacy of a synthetic peptide vaccine against Semliki Forest virus.

The antibody response to a previously defined B-cell epitope of Semliki Forest virus (SFV) was investigated in male BALB/c (H-2d) mice. The B-cell epitope, located at amino acid positions 240 to 255 of the E2 protein, was linked to an H-2d-restricted T-helper cell epitope of SFV located at positions 137 to 151 of the E2 protein. Colinearly synthesized peptides, of either T-B or B-T polarity, mixed with different adjuvants (the nonionic block copolymer L 180.5, a water-oil-water [W/O/W] emulsion of L 180.5, Montanide, and Q VAC) were used for immunization. Generally, after one booster immunization, high serum antibody titers were measured against either peptide. With Q VAC and W/O/W L 180.5 as adjuvants, the titers of SFV-reactive (nonneutralizing) antibodies were consistently much higher after immunization with the T-B peptide than with the B-T peptide, which was reflected in a higher vaccine efficacy. With these two adjuvants, the survival ratio in T-B peptide-immunized mice was 82%, compared with 8% in B-T peptide-immunized mice. Intermediate results were obtained with the adjuvant Montanide. L 180.5 alone was ineffective in this study. All immunoglobulin G (IgG) isotypes were induced with either adjuvant, but Q VAC was clearly the most effective in inducing IgG2a and IgG2b isotypes with the T-B peptide as the antigen. Subsequently, monoclonal antibodies (MAbs) of IgM, IgG1, IgG2a, IgG2b, and IgG3 subclasses were prepared against the B-cell epitope. These nonneutralizing but SFV-reactive MAbs protected 40 to 80% of mice against a lethal challenge with SFV. Control mice all died. The availability of those antipeptide MAbs allowed competition binding assays with a previously characterized panel of E2-specific MAbs. Binding of enzyme-labeled antipeptide MAbs was very effectively inhibited by two strongly SFV-neutralizing mutually competitive MAbs, suggesting that the linear B-cell epitope (amino acids 240 to 255) is associated with a major neutralization site of SFV.

Detection of a shared idiotope on two encephalomyocarditis virus neutralizing monoclonal antibodies by neutralization inhibition enzyme immunoassay.

Idiotypic cross-reactivity between encephalomyocarditis virus (EMCV) neutralizing monoclonal antibodies UM 21.1 (IgG2b) and UM 21.3 (IgG2a) was detected by neutralization inhibition enzyme immunoassay using polyclonal and monoclonal anti-idiotypic antibodies. One strongly cross-reactive anti-idiotypic monoclonal antibody, designated 21.1A5 (IgG2b), might recognize a recurrent idiotope on EMCV neutralizing antibodies but it did not induce EMCV neutralizing anti-anti-idiotypic antibodies in homologous BALB/c mice.