Experimental evolution of external immune defences in the red flour beetle.

The red flour beetle, Tribolium castaneum, secretes quinones that control the microbial flora in the surrounding environment. These secretions act as an external immune defence that provides protection against pathogens. At high concentrations, however, these secretions are harmful to the host itself, and selection may thus have optimized the level of expression under natural conditions. Here, we show that the expression of external immunity responded to selection during experimental evolution within a few generations. At the same time, one component of internal immune defence (phenoloxidase activity) was compromised in beetles selected for either high or low external defences. Intriguingly, offspring protection against a natural pathogen was reduced in flour obtained from beetle lines selected for low amounts of secretions. Altogether, this suggests that external and internal immune defences work together efficiently under natural conditions, whereas every manipulation on the side of external immune defence comes with costs to the internal immune defence.

Description of Hymenobacter arizonensis sp. nov. from the southwestern arid lands of the United States of America.

Strain OR362-8(T) was isolated from a biological soil crust sample collected from the southwestern arid lands of the United States of America, using BG11-PGY medium. Cells of OR362-8(T) were found to be rod shaped; occur singly, as pairs and in groups; non-motile; positive for catalase, oxidase, phosphatase and gelatinase; hydrolyze starch; contain iso-C(15:0), anteiso-C(15:0), iso-C(15:1)G, C(16:1ω5c) and summed feature 3 (C(16:1(ω7c))/iso-C(15:0) 2OH as defined by the MIDI system) as the major fatty acids; and MK-7 as the sole respiratory quinone. A BLAST sequence similarity search using 16S rRNA gene sequence of OR362-8(T) identified Hymenobacter as the nearest genus with a similarity of 90.4-96.9 %. The phylogenetic analyses based on the phenetic methods UPGMA, NJ, ME and DNA parsimony resulted in the clustering of OR362-8(T) with Clade 1 Hymenobacter species represented by Hymenobacter glaciei, Hymenobacter antarcticus, Hymenobacter flocculans, Hymenobacter metalli and Hymenobacter soli with the closest being the Hymenobacter glaciei (96.9 % 16S rRNA gene sequence similarity). Besides the strong phylogentic affiliation, OR362-8(T) also exhibited significant phenotypic and chemotaxonomic differences with the members of Clade 1 Hymenobacter spp. More importantly, the DNA G+C content (mol%) of OR362-8(T) is very high (70 %) compared to the nearest species identified by phylogenetic analysis. Based on the phylogenetic, phenotypic and chemotaxonomic characteristics, OR362-8(T) was assigned to a novel species for which we propose here the name Hymenobacter arizonensis sp. nov., with OR362-8(T) (=ATCC BAA 1266(T) = DSM 17860(T) = JCM 13504(T)) as the type strain.

Immunoconjugates of geldanamycin and anti-HER2 monoclonal antibodies: antiproliferative activity on human breast carcinoma cell lines.

BACKGROUND: HER2 is a membrane receptor whose overexpression is strongly associated with poor prognosis in breast carcinomas. Inhibition of HER2 activity can reduce tumor growth, which led to the development of Herceptin, an anti-HER2 monoclonal antibody (MAb) that is already in clinical use. However, the objective response rate to Herceptin monotherapy is quite low. HER2 activity can also be inhibited by the highly cytotoxic antibiotic geldanamycin (GA). However, GA is not used clinically because of its adverse toxicity. Our purpose was to enhance the inhibitory activity of anti-HER2 MAb by coupling it to GA. METHODS: We synthesized 17-(3-aminopropylamino)GA (17-APA-GA) and conjugated it to the anti-HER2 MAb e21, to form e21 : GA. The noninternalizing anti-HER2 MAb AE1 was used as a control. Internalization assays and western blot analyses were used to determine whether the anti-HER2 MAbs and their immunoconjugates were internalized into HER2-expressing cells and reduced HER2 levels. All statistical tests were two-sided. RESULTS: The immunoconjugate e21 : GA inhibited the proliferation of HER2-overexpressing cell lines better than unconjugated e21 (concentration required for 50% inhibition = 40 versus 1650 microg/mL, respectively). At 15 microg/mL, e21 : GA reduced HER2 levels by 86% within 16 hours, whereas unconjugated e21, 17-APA-GA, or AE1 : GA reduced HER2 levels by only 20%. These effects were not caused by release of 17-APA-GA from the immunoconjugate because immunoconjugates containing [(3)H]GA were stable in serum at 37 degrees C. Furthermore, e21 : GA did not significantly inhibit proliferation of the adult T-cell leukemia cell line HuT102, which is HER2 negative yet highly sensitive to GA. CONCLUSIONS: Our findings suggest that conjugating GA to internalizing MAbs enhances the inhibitory effect of the MAbs. This approach might also be applied in cellular targeting via growth factors and may be of clinical interest.

Mechanism of allergic cross-reactions--I. Multispecific binding of ligands to a mouse monoclonal anti-DNP IgE antibody.

A recently developed solid-phase binding assay was used to investigate the specificity of ligand binding to a mouse monoclonal anti-dinitrophenyl IgE [IgE(aDNP)]. All DNP-amino acids, that were tested, inhibited the binding of radio-labeled IgE(aDNP) to DNP covalently attached to polystyrene microtiter plates; however, the concentration for 50% inhibition varied within four orders of magnitude, DNP-L-serine being the most, DNP-proline the least potent inhibitor. In addition to DNP analogues a large number (2074) of drugs and other compounds were tested for their ability to compete with DNP for the binding site of IgE(aDNP). At the concentrations used for screening 59% of the compounds had no significant inhibition; 19% inhibited the binding of IgE(aDNP) more than 50%. Several families of compounds (tetracyclines, polymyxines, phenotiazines, salicylates and quinones) of effective competitors were found. Within these families change in the functional groups attached to the "family stem" had major effects on the affinity of ligand binding. The occurrence frequencies of interactions of ligands with IgE(aDNP) is in good agreement with a semi-empirical model for multispecific antibody-ligand interactions.

A systematic search for structure-activity relationships of skin contact sensitizers: methodology.

A computerized resource for the systematic evaluation of the structure-activity relationships and other aspects of contact allergens is described. This resource consists of a data base of results of contact dermatitis tests and a structural classification scheme for contact allergens that is called a Structure-Activity (S/A) Tree. The data base now contains approximately 2200 test results extracted from the journal Contact Dermatitis (1975-1982) and is continually being expanded. The S/A Tree is being developed to provide an index to structure-activity relationships of contact allergens; 63 structural groups are currently indexed. Analyses of benzoquinones and gallic acid esters are presented as examples of the potential application of this resource to such problems as the identification of potential cross-reactants, appropriate test concentrations and vehicles, and the reliability of available test results.

Monoclonal anti-histamine antibody. Preparation, characterization and application to enzyme immunoassay of histamine.

An enzyme immunoassay to measure histamine has been developed. A histamine-bovine serum albumin conjugate was prepared using 1,4-benzoquinone as the coupling agent and was employed to immunize mice for the preparation of monoclonal antibodies against histamine. After an initial screening to identify antigen-binding monoclonal antibodies the clones were isolated by limiting dilution cloning, grown in ascites and antibodies which had been secreted into the ascitic fluid were precipitated by ammonium sulphate at 50% saturation. A systematic approach for the determination of epitope specificities of monoclonal antibodies was performed. It was found that for the most specific antibody the main epitope encompassed the 2-histaminyl-1,4-benzoquinone moiety and that the KD value determined by indirect ELISA was 1.5 X 10(-8) M for the hapten part of the immunogen and 4.6 X 10(-10) M for a histamine-Bq-ovalbumin conjugate. The selected monoclonal antibody could not recognize histidine or methyl-histamine. Using this antibody, we developed an enzyme immunoassay for histamine and pg amounts could be detected. The same assay was used to quantify the allergic release of histamine from guinea pig lung mast cells. Results obtained either by the present enzyme immunoassay or by a fluorometric assay were closely correlated (correlation coefficient r = 0.9702, n = 37).

The cytotoxic activity of heterologous anti-L-1210 antibodies conjugated with quinones.

Biological effects of conjugates of quinone derivatives with antibodies were studied. Two methods of conjugation of aziridine derivatives of quinone with anti-L-1210 antibodies were used: the direct substitution of the protein amino groups or thiolation of immunoglobulins and subsequent substitution of the sulfhydryl groups introduced. The reactions were carried out in 10% solutions of DMF at pH 8.3-8.9. The biological activity of conjugates, in vitro, was controlled using L-1210 and HeLa cells by measuring the inhibition of incorporation of labeled thymidine. The growth of L-1210 cells was more strongly inhibited by conjugates prepared with anti-L-1210 antibodies than by conjugates with nonimmune IgG. No difference in biological activity was found between conjugates prepared from anti-L-1210 antibodies and normal IgG in tests with HeLa cells.

Cancer morbidity in rheumatoid arthritis: role of estrogen metabolites.

Estrogen metabolites have been implicated in rheumatoid arthritis (RA) and cancer, although the mechanism remains unestablished. Some estrogen metabolites, which are used for the assessment of cancer risk, play an important role in RA. The pathways by which malignancies associated with RA remain elusive. Possible mechanism involves enzymatic or nonenzymatic oxidation of estrogen into catecholestrogen metabolites through semiquinone and quinone redox cycle to produce free radicals that can cause DNA modifications. Modifications of DNA alter its immunogenicity and trigger various immune responses leading to elevated levels of cancer and RA antibodies. However, the role of different estrogen metabolites as a mediator of immune response cannot be ruled out in various immune-related diseases.

Generation of a novel anti-geldanamycin antibody.

Geldanamycin (GA) and herbimycin A are benzoquinone ansamycins (BAs) that inhibit the molecular chaperone HSP90. The central role of HSP90 in maintaining the conformation, stability, and function of key oncogenic proteins involved in signal transduction pathways renders BAs attractive candidates for clinical development. Two GA derivatives, 17-allylamino-17-demethoxygeldanamycin and 17-demethoxy-17-N,N-dimethylaminoethylamino-geldanamycin are currently evaluated in clinical trials. The present study demonstrates generation of a polyclonal antibody elicited against GA that was conjugated to keyhole limpet hemocyanin via its 17 position. The anti-GA antibody recognizes GA as well as other BAs, suggesting its possible application for monitoring plasma levels of GA derivatives. The specificity of the antibody towards BAs is demonstrated by its inability to recognize radicicol, an HSP90 inhibitor not related to BAs. This antibody thus presents a novel research tool as well as a possible alternative approach for monitoring drug levels in patients.

Allergic contact dermatitis caused by naturally occurring quinones.

Quinones play a major role in allergic contact dermatitis caused by plants. The principal allergens are benzoquinones or naphthoquinones but also compounds, such as catechols and other phenolic or flavonoid compounds, which are bioconverted into ortho-quinones or para-quinones. The high electrophilic reactivity of these compounds toward nucleophilic residues of proteins associated with lipophilic properties may explain that they are strong sensitizers. The more important allergens are reported and their structure-activity relationship is discussed.

Sensitizing capacity of naturally occurring quinones. V. 2.6-dimethoxy-p-benzoquinone: occurrence and significance as a contact allergen.

2.6-dimethoxy-1,4-benzoquinone has already been discovered in more than 25 different plants and woods. Several authors have shown its strong bacteriostatic activity against micro-organisms. In 1972 a positive skin reaction to 2.6-dimethoxybenzoquinone was obtained in a patient allergic to Sucupira wood (Bowdichia nitida Benth.). This compound could be isolated from the wood. During the chemical investigations of other commercial woods which have been described as the cause of allergic contact dermatitis, 2.6-dimethoxybenzoquinone could be isolated from an additional 21 different species. In the case of Australian blackwood Acacia melanoxylan R.BR., its structure was elucidated by x-ray analysis. Sensitization of guinea pigs revealed that 2.6-dimethoxybenzoquinone is a relatively good sensitizer. In some of the woods investigated, quinones have never been discovered before, for example in Makoré, Australian blackwood, Wengé, White wood, Afrormosia and Afzelia. In 10 of them only this quinone was detectable. Besides its allergenic properties, 2.6-dimethoxybenzoquinone may be responsible for the high termite resistance of these woods. In all cases of contact dermatitis from these wood species in which quinoid allergens other than 2,6-dimethoxybenzoquinone could be detected, this quinone should be used for patch tests.

Investigation of the prohapten concept. Cross reactions between 1,4-substituted benzene derivatives in the guinea pig.

It has been proposed that the cross-reactions seen clinically between hydroquinone and para-phenylenediamine (PPD) arise from the formation of a common hapten, benzoquinone, in vivo, and that these chemicals therefore represent "prohaptens". A series of 1,4-substituted benzene derivatives has been used to examine this prohapten concept in the guinea pig model. Using both topical and intradermal routes of application, it is demonstrated that in the guinea pig 1,4-substituted benzene derivatives capable of oxidation to benzoquinone, including hydroquinone and PPD, show only restricted evidence of cross-reactions. These results support the prohapten concept. However taken in combination with data on cross-reactivity with 1,2- and 1,3-substituted benzenes, rather than giving rise to a single common hapten, they can be more readily interpreted as the formation of a spectrum of antigenic determinants in vivo, some of which are shared in common.

Influence of chemical reactivity of urushiol-type haptens on sensitization and the induction of tolerance.

Contact sensitization to components of the urushiol oils of poison oak and poison ivy appears to require covalent bond formation between the o-quinones derived from urushiol catechols and nucleophilic groups on proteins. Previous studies using a murine delayed hypersensitivity model demonstrated that 5-methyl-3-pentadecylcatechol (5-Me-PDC) is an epicutaneous tolerogen to the parent compound and a weak sensitizer to itself. To investigate further the structural requirements for sensitization vs suppression, 5,6-dimethyl-3-pentadecylcatechol (5,6-di-Me-PDC) and 4,5,6-trimethylpentadecylcatechol (4,5,6-tri-Me-PDC) were synthesized. The former compound is blocked at both preferred sites for covalent bond formation and the latter is completely blocked towards conjugate addition reactions. These compounds were tested for sensitizing and suppressive ability. Epicutaneous application of both analogs suppressed subsequent induction of sensitization to 3-pentadecylcatechol (PDC) and 3-heptadecylcatechol (HDC). Lymph node cells from animals treated with 5,6-di-Me-PDC could transfer suppression. The dimethyl analog, 5,6-di-Me-PDC, but not the trimethyl analog also exhibited weak sensitizing capacity. The urushiol analogs 5-pentadecylresorcinol (PDR) and 3-heptadecylveratrole (HDV) which cannot form o-quinones were found to be ineffective sensitizers as well. HDV in addition produced no blastogenesis in draining lymph nodes whereas lymph node cell proliferation induced by 4,5,6-tri-Me-PDC followed the same kinetics as previously observed for HDC. PDR elicited weak proliferation with a different time course. These and previous studies indicate that blocking the C5-position on the catechol ring favors the induction of suppression, although some sensitizing capacity may be retained. Covalent bond formation may not be necessary for the induction of active suppressor cell populations.

[Isolation and study of the properties of specific antisera to [leu]enkephalin]. / Poluchenie i izuchenie svoistv spetsificheskikh antisyvorotok k [leu]énkefalinu.

The immunogenic conjugates of [leu]enkephalin and bovine serum albumin (BSA) with bisdiazobenzidine and 1,4-benzoquinone as bifunctional reagents have been synthesized. The antisera with high titer of antibodies to [leu]enkephalin have been obtained at rabbit [correction of rat] immunization by both conjugates. The antiserum obtained at immunization by conjugate [leu]enkephalin-benzoquinone-BSA possesses the high affinity and sensitivity to [leu]enkephalin.