Ab Complex Research Articles

Solution equilibria of some quaternary metal complexes involving iminodiacetic acid and L-pyrrolidine-2-carboxylic acid

Equilibrium based computer studies on quaternary complex systems, viz. Mn II /Co II /Pb II iminodiacetic acid (IMDA = A 2- ) and L-pyrrolidine-2-carboxylic acid (proline = B - ) systems demonstrate the formation of mixed-metal mixed-ligand complexes of stoichiometry M I M 2 AB and M I M 2 A 2 B in addition to MAB, M 2 A 2 B, MA and MB complexes for all metal ions. Formation constants of the complexes at 35 ± 1 °C and at fixed ionic strength, I = = 0.1 mol dm -3 (NaNO 3 ) were evaluated using SCOGS computer program and the complex formation equilibria were elucidated with the aid of speciation curves. Stability order of metal complexes and solution structure of hetrobinuclear M 1 M 2 AB complexes have been discussed.

Hemextin AB Complex, a Unique Anticoagulant Protein Complex from Hemachatus haemachatus (African Ringhals Cobra) Venom That Inhibits Clot Initiation and Factor VIIa Activity

During injury or trauma, blood coagulation is initiated by the interaction of factor VIIa (FVIIa) in the blood with freshly exposed tissue factor (TF) to form the TF.FVIIa complex. However, unwanted clot formation can lead to death and debilitation due to vascular occlusion, and hence, anticoagulants are important for the treatment of thromboembolic disorders. Here, we report the isolation and characterization of two synergistically acting anticoagulant proteins, hemextins A and B, from the venom of Hemachatus haemachatus (African Ringhals cobra). N-terminal sequences and CD spectra of the native proteins indicate that these proteins belong to the three-finger toxin family. Hemextin A (but not hemextin B) exhibits mild anticoagulant activity. However, hemextin B forms a complex (hemextin AB complex) with hemextin A and synergistically enhances its anticoagulant potency. Prothrombin time assay showed that these two proteins form a 1:1 complex. Complex formation was supported by size-exclusion chromatography. Using a "dissection approach," we determined that hemextin A and the hemextin AB complex prolong clotting by inhibiting TF.FVIIa activity. The site of anticoagulant effects was supported by their inhibitory effect on the reconstituted TF.FVIIa complex. Furthermore, we demonstrated their specificity of inhibition by studying their effects on 12 serine proteases; the hemextin AB complex potently inhibited the amidolytic activity of FVIIa in the presence and absence of soluble TF. Kinetic studies showed that the hemextin AB complex is a noncompetitive inhibitor of soluble TF.FVIIa amidolytic activity, with a Ki of 50 nm. Isothermal titration calorimetric studies showed that the hemextin AB complex binds directly to FVIIa with a binding constant of 1.62 x 10(5) m(-1). The hemextin AB complex is the first reported natural inhibitor of FVIIa that does not require a scaffold to mediate its inhibitory activity. Molecular interactions of the hemextin AB complex with FVIIa/TF.FVIIa will provide a new paradigm in the search for anticoagulants that inhibit the initiation of blood coagulation.

Immune Complex-Dependent Remodeling of the Airway Vasculature in Response to a Chronic Bacterial Infection

Chronic inflammation in the airways is associated with dramatic architectural changes in the walls of the airways and in the vasculature they contain. In this study, we show that the adaptive immune system is essential for airway remodeling that occurs in mice that are chronically infected with the respiratory pathogen Mycoplasma pulmonis. Angiogenesis, lymphangiogenesis, and epithelial remodeling were greatly reduced in mice that lacked B cells. Substantiating a role for Ab and airway immune complexes, we found that the transfer of immune serum to B cell-deficient mice could reconstitute pathogen-induced angiogenesis. Inflammatory cells recruited to the infected airways were activated by the humoral response, and this activation correlated with the induction of genes for remodeling factors such as vascular endothelial growth factor-D. The results reveal a novel pathway whereby T cell-dependent humoral immunity to a persistent airway infection can induce inflammation-dependent angiogenesis, lymphangiogenesis, and chronic airway pathology.

Direct observation of UV-B radiation effect on antigen–antibody coupling using surface plasmon resonance

We studied the effect of UV irradiation on (non)specific bounding of bovine serum albumin (BSA) and mouse antiBSA immunoglobulin (IgG) using a surface plasmon resonance (SPR) apparatus Biosuplar-2 and special photodetectors. It was found that specific bounding of AG–AB complex is broken under UV irradiation. Contrary to this, UV irradiation of BSA layer before its contact with IgG did not demonstrate any peculiarities. On the basis of quantitative determination of kinetics of specific bounding and washing and precise measurement of photon flux, the product γσ of quantum yield γ and active centre cross-section σ was evaluated to be about 10 −20 cm 2. As the dimension of amino acid residues tryptophan and tyrosine is about 1 nm, we have concluded that the quantum yield of reaction is very small (about 0.001). This indicates at presence of a barrier for direct photochemical reaction between the interacting substances.

Calcineurin B protects calcineurin A against denaturation by urea

Calcineurin (CN), a heterodimer composed of a catalytic subunit, calcineurin A (CNA) and regulatory subunit, calcineurin B (CNB), is involved in many cellular processes. We investigated the denaturation of CNA by urea in the presence or absence of CNB and found that CNB protected CNA against urea. The phosphatase activity of CNA that had been exposed to low urea concentrations (below 4 M), in the presence CNB, was higher than that of the separately urea-treated subunits mixed just prior to assay. In order to analyze the protection of CNA by CNB, we investigated the K m and V max, and intrinsic fluorescence, of CNA that had been exposed to various concentrations of urea in the presence or absence of CNB. CN had an increased V max and decreased K m when exposed to 1 to 2 M urea. In addition, the kinetic parameters and intensity of intrinsic fluorescence of the AB complex and isolated subunits were quite different in 3 M urea. These results indicate that CNB not only plays an important role in regulating CNA, but also protects it against denaturation by urea.

Analysis of secretory immunoglobulin A in human saliva by laser-induced fluorescence capillary electrophoresis

The utility of capillary electrophoresis (CE) has been demonstrated for the analysis of secretory immunoglobulin A (sIgA) in human saliva. The amount of sIgA in saliva correlates with immune status. For detecting salivary sIgA, laser-induced fluorescence was conducted in this report for signal amplification. sIgA and anti-sIgA antibody were labeled with cyanine fluorescence (Cy5) for competitive immunoassay and non-competitive analysis, respectively. Cy5 was excited by He–Ne laser with a wavelength of 635 nm, with maximum emission at 670 nm. Migration time during electrophoresis depended on whether sIgA–Cy5 was mixed with antibody or anti-sIgA–Cy5 mixed with sIgA to form Ag–Ab complex. The results indicated that CE competitive immunoassay was effective for analyzing serum sIgA, but not for salivary sIgA. However, salivary sIgA can be analyzed by complex formation assay. The peak area of the complex was proportional to the amount of sIgA added. A standard linear regression curve was generated using purified sIgA. From this standard curve, the amount of sIgA from saliva of either normal or immunocompromised patients can be calculated from the Ag–Ab complex peak area.

A systematic spectroscopic study of four bimetallic thiocyanates of chemical formula AB(SCN)4: ZnCd(SCN)4 and AHg(SCN)4 (A=Zn, Cd, Mn) as UV nonlinear optical crystal materials

Bimetallic thiocyanate complexes of chemical formula AB(SCN)4(ABTC): ZnCd(SCN)4 and MHg(SCN)4 (M=Zn, Cd, Mn) were discovered as potentially useful crystal materials in ultraviolet second-harmonic generation. The structures of these compounds were derived from powder X-ray diffraction data, and unit-cell parameters were calculated by the TREOR program. Their vibrational spectra were studied by Raman spectroscopy, which show that the characteristic vibrational modes of AB(SCN)4 crystals arise mainly from the internal vibrations of the SCN, A(NCS)4, and B(SCN)4 clusters. The reason of the high optical nonlinearities of ABTC crystals has been studied by the use of the Raman spectra and the molecular orbital theory: the distorted A(NCS)4 and B(SCN)4 tetrahedra and the conjugated charge-transfer (–A–NCS–B–) bridges that connect all the distorted tetrahedra together.

Capillary electrophoretic enzyme immunoassay with electrochemical detection for thyroxine

A capillary electrophoretic enzyme immunoassay with electrochemical detection (CE-EIA-ED) has been developed. In this method, antigen (Ag) competes with horseradish peroxidase (HRP)-labeled antigen (HRP–Ag) for a limited number of antibody (Ab) binding sites. The free HRP–Ag and the bound HRP–Ag–Ab complex are separated by capillary electrophoresis in a separation capillary. Then they catalyze the oxidation of their enzyme substrate 3,3 ′,5,5 ′-tetramethylbenzide (TMB (reduced form)) with H 2O 2 in a reaction capillary, which follows the separation capillary. The reaction product (TMB (oxidized form)) is amperometrically determined using a carbon fiber microdisk bundle electrode at the outlet of the reaction capillary. Due to the amplification of the enzyme, the concentration of TMB(Ox) is much higher than those of free HRP–Ag and the bound HRP–Ag–Ab complex. Therefore, the limit of detection (LOD) of CE-EIA-ED is very low. The method has been used to determine thyroxine in human serum. A concentration of LOD of 3.8×10 −9 mol/L, which corresponds to a mass LOD of 23.2 amol, was achieved.

Liposomes Targeted to Fc Receptors for Antigen Presentation by Dendritic Cells In Vitro and In Vivo∗

The adaptive immune responses of mammals depend on cells specialized in the acquisition of antigen (Ag), called Ag presenting cells (APC), and cells that respond to that Ag, T and B lymphocytes. The two major arms of the adaptive immune response are antibody (Ab) production and cytotoxicity. T lymphocytes are divided into CD4+ and CD8+ T cells. CD4+ T cells are necessary to induce the immunoglobulin gene rearrangements necessary for the production of somatically mutated IgG Ab by B cells. Cytotoxicity is primarily a property of CD8+ T cells, but it requires CD4+ T-helper cells for complete differentiation and acquisition of effector functions. The induction of the immune response is thus a complex series of sequential interactions involving Ag contact with APC, APC contact with lymphocytes, and interaction among lymphocytes. Liposomes are very simple and lack microbial elements designed to inhibit immune responses and elements, such as endotoxin, repeated carbohydrates, and nucleic acids. CD4+ T cells do not recognize protein Ag as such but recognize Ag after uptake, partial proteolysis, and re-expression of Ag-derived peptides at the surface of APC in association with major histocompatibility complex (MHC)-encoded class II molecules. APC include dendritic cells (DC), macrophages, and B cells. DC is widely distributed in tissues, and both DC and macrophages use antibody secreted by B cells to bind Ag for enhanced presentation to naive CD4 T cells. DC and macrophages have receptors for Ag–Ab complexes, the immunoglobulin Fc receptors, which are a family of molecules at the surface of DC.

Efeitos das respostas de observação diferenciais sobre a aprendizagem de relações condicionais com estímulos complexos

Este estudo avaliou se relações entre os componentes do estímulo modelo complexo exerceriam controle condicional em tarefas de matching-to-sample simultâneo. Na Fase 1, 3 crianças com necessidades especiais de ensino foram expostas ao treino das relações A1B1 e A2B2 e ao teste das respectivas relações simétricas. Em seguida, as contingências de treino exigiram respostas de observação diferenciais que consistiram no estabelecimento de relações condicionais de identidade entre estímulos complexos (relações AB-AB) precedendo o acesso ao treino das relações condicionais ABX. Neste treino, diante de estímulos modelos complexos cujos componentes sustentavam condicionalidade treinada (A1B1 e A2B2), X1 foi o estímulo de escolha correto; X2 exerceu esta função quando os componentes do estímulo modelo não sustentavam tal relação (A1B2 e A2B1). Na Fase 2, ocorreria o treino PQ, testes QP e PQX que avaliariam a extensão do controle condicional definido pelas relações entre os estímulos P e Q. As três crianças registraram a aprendizagem das relações AB, a emergência das relações simétricas e índices elevados de acerto nas respostas de observação diferenciais, ou seja, no estabelecimento das relações condicionais de identidade com estímulos complexos. Contudo, as três demonstraram relações de controle distintas das previstas no treino ABX, sendo, portanto, o experimento finalizado na Fase 1. Tais resultados sugerem uma independência funcional entre as habilidades discriminativas exigidas nas duas contingências de ensino de relações condicionais com estímulos modelo complexos.

Antibody targeted gene transfer to endothelium.

One of the drawbacks of the currently available vectors for gene therapy is the lack of selectivity in gene delivery. We have therefore investigated a strategy to generate immunoliposomes to target non-viral vectors to cell surface receptors on endothelium. We have developed a novel method of coupling antibodies (Abs) to liposomes complexed to DNA, using mild heat treatment to aggregate the immunoglobulin G (IgG). The interaction of plasmid DNA, liposomes and Abs was measured using a gel retardation assay and a resonant mirror biosensor. The size of the transfection complex was determined by light scattering, and the binding and internalization of the complex to cells was followed using flow cytometry. The transfection ability was tested on cell lines and primary cells in vitro and human corneal or vascular tissues ex vivo. The interaction of antibodies with liposomes is relatively stable (t(1/2) congruent with 45 min). The size of the liposome, Ab and DNA complex was found to be around 500 nm in 4% BSA. The addition of anti-transferrin receptor Abs increased the internalization of the liposome-DNA complex into cells. Abs against both transferrin receptor and E-selectin were shown to augment transfection efficiency of liposomes to cell expressing the appropriate antigens. They are also shown to be efficient in mediating gene delivery to corneal and vascular tissues ex vivo. We have shown that our novel vector is capable of in vitro and ex vivo gene delivery to cells and human tissues including cornea, artery and vein. In particular, an Ab against E-selectin was effective at selectively delivering genes to activated endothelial cells expressing the adhesion molecule. Such a strategy will have applications for targeting these tissues prior to transplantation or autologous grafting, and, in the longer term, may allow in vivo targeting of gene therapy to inflammatory sites.

Positive selection of self-MHC-reactive T cells by individual peptide-MHC class II complexes.

If T cells require specific interactions with MHC-bound peptides during positive selection, then the specificities of T cells selected by one peptide should be distinct from those selected by another. We have examined positive selection of CD4 T cells in four strains of mice, each overexpressing a different peptide-1-A(b)(A(b)) complex. We show that a subset of CD4 T cells is selected by the overexpressed peptide and that the specificities of the CD4 T cells, as measured by reactivity to wild-type antigen-presenting cells, vary greatly depending on which peptide is overexpressed. These differences in specificity are mediated through positive selection not negative selection. Each of the four peptide-A(b) complexes appears to adopt a different conformation, and these differences correlate with the differences in reactivity. Our results suggest that individual peptide-MHC complexes positively select different subsets of self-MHC-reactive T cells and that the conformation of the peptide-MHC complex may contribute to this process.

IL-4 and T cells are required for the generation of IgG1 isotype antibodies against cardiolipin.

Infection with Mycobacterium tuberculosis induces Abs against a vast array of mycobacterial lipids and glycolipids. One of the most prominent lipid Ags recognized is cardiolipin (CL). The kinetics of the generation of anti-CL Abs during infection reveals that IgM titers to CL increase over time. Interestingly, at day 30 postinfection CL-specific IgG1 appears, an isotype usually dependent on T cell help. Using an immunization schedule with CL/anti-CL Ab complexes, which induces antiphospholipid syndrome in mice, we show that the generation of IgG1 to CL requires IL-4 and that optimal production is T cell dependent. IgG1 production to CL was impaired in nude (nu/nu) mice devoid in conventional T cells, but was not affected in mice deficient for either alphabeta TCR(+), gammadelta TCR(+), CD4(+), CD8(+), or NK1.1(+) T cells. We conclude that IgG1 production to CL depends on T cell help and IL-4, which can be provided by different T cell populations. This is the first report that IL-4 is indispensable for the induction of IgG1 Abs to lipid Ags.

A negative-selection strategy for depleting myeloma cells from patients' BM and/or leukapheresis blood

Autologous transplantation improves survival in multiple myeloma patients, however, most eventually relapse. As an attempt towards improving relapse-free survival, we designed a negative-selection purging strategy, to remove myeloma cells from leukapheresis harvests using MAbs specific for Ags on myeloma cells. CD38 is highly expressed on myeloma plasma cells, but expressed at lower levels on normal progenitors and absent on in vivo repopulating cells. We evaluated depletion of CD38-expressing cells, with or without depletion of B-cell Ag-expressing cells. Using myeloma BM or blood cells diluted into allogeneic G-CSF primed leukapheresis cells, bispecific tetrameric Ab complexes that bind dextran iron particles were used to label and retain cells in a magnetic column, StemSep. Depletion efficacy was measured by semi-quantitative allele-specific oligonucleotide (ASO)-PCR amplification of patients' clonotypic IgH gene. Low (0.2 microg/mL) concentrations of anti-CD38 with CD19 and CD20 complexes depleted approximately 3-5 logs of clonotypic cells, with recovery of approximately 19% of colony-forming cells, approximately 50% primitive progenitors measured by LTCIC and retention of non-obese diabetic /SCID engrafting ability. Scale-up experiments using leukapheresis harvests and 0.5-1 x 10(10) cell capacity columns demonstrated no loss of log depletion of highly positive cells, or recovery of unlabelled cells. These results compare favorably with other purging techniques and allow the retention of most normal BM cells, including T cells, which may be important for immunity. These results support the development of a clinical trial using this strategy for purging myeloma cells.

Anti-Double-Stranded DNA Antibodies and Immunostimulatory Plasmid DNA in Combination Mimic the Endogenous IFN-α Inducer in Systemic Lupus Erythematosus

AbstractPatients with systemic lupus erythematosus (SLE) have increased blood levels of IFN-α, which correlate to disease activity. We previously identified an IFN-α-inducing factor (IIF) in the blood of SLE patients that activated the natural IFN-α-producing cells in cultures of normal PBMC. The SLE-IIF contained DNA and IgG, possibly as small immune complexes. In our study, we demonstrated that SLE-IIF correlated to the presence of anti-dsDNA Abs in patients and contained anti-dsDNA Abs as an essential component. Purified anti-DNA Abs or SLE-IgG caused only a weak IFN-α production in cultures of normal PBMC in the presence of costimulatory IFN-α2b. However, they converted the plasmid pcDNA3, which itself induced no IFN-α production in PBMC, into an efficient IFN-α inducer. A human monoclonal anti-ss/dsDNA Ab had the same effect. This IFN-α-inducing activity of the plasmid was abolished by methylation, suggesting that unmethylated CpG DNA motifs were important. Like IIF in SLE serum, the combination of SLE-IgG and pcDNA3 appeared to stimulate IFN-α production in natural IFN-α-producing cells, a unique cell population resembling immature dendritic cells. The IFN-α production was greatly enhanced by IFN-α2b and IFN-β, and for SLE-IIF it was also enhanced by GM-CSF but inhibited by IL-10. We have therefore identified a new function of DNA-anti-DNA Ab complexes, IFN-α induction, that might be important in the pathogenesis of SLE.

Mutants of Escherichia coli heat-labile toxin act as effective mucosal adjuvants for nasal delivery of an acellular pertussis vaccine: differential effects of the nontoxic AB complex and enzyme activity on Th1 and Th2 cells.

Mucosal delivery of vaccines is dependent on the identification of safe and effective adjuvants that can enhance the immunogenicity of protein antigens administered by nasal or oral routes. In this study we demonstrate that two mutants of Escherichia coli heat-labile toxin (LT), LTK63, which lacks ADP-ribosylating activity, and LTR72, which has partial enzyme activity, act as potent mucosal adjuvants for the nasal delivery of an acellular pertussis (Pa) vaccine. Both LTK63 and LTR72 enhanced antigen-specific serum immunoglobulin G (IgG), secretory IgA, and local and systemic T-cell responses. Furthermore, using the murine respiratory challenge model for infection with Bordetella pertussis, we demonstrated that a nasally delivered diphtheria, tetanus, and acellular pertussis (DTPa) combination vaccine formulated with LTK63 as an adjuvant conferred a high level of protection, equivalent to that generated with a parenterally delivered DTPa vaccine formulated with alum. This study also provides significant new information on the roles of the binding and enzyme components of LT in the modulation of Th1 and Th2 responses. LTK63, which lacks enzyme activity, promoted T-cell responses with a mixed Th1-Th2 profile, but LTR72, which retains partial enzyme activity, and the wild-type toxin, especially at low dose, induced a more polarized Th2-type response and very high IgA and IgG antibody titers. Our findings suggest that the nontoxic AB complex has broad adjuvant activity for T-cell responses and that the ADP-ribosyltransferase activity of the A subunit also appears to modulate cytokine production, but its effect on T-cell subtypes, as well as enhancing, may be selectively suppressive.

Presentation of antigen in immune complexes is boosted by soluble bacterial immunoglobulin binding proteins.

Using a snake toxin as a proteic antigen (Ag), two murine toxin-specific monoclonal antibodies (mAbs), splenocytes, and two murine Ag-specific T cell hybridomas, we showed that soluble protein A (SpA) from Staphylococcus aureus and protein G from Streptococcus subspecies, two Ig binding proteins (IBPs), not only abolish the capacity of the mAbs to decrease Ag presentation but also increase Ag presentation 20-100-fold. Five lines of evidence suggest that this phenomenon results from binding of an IBP-Ab-Ag complex to B cells possessing IBP receptors. First, we showed that SpA is likely to boost presentation of a free mAb, suggesting that the IBP-boosted presentation of an Ag in an immune complex results from the binding of IBP to the mAb. Second, FACS analyses showed that an Ag-Ab complex is preferentially targeted by SpA to a subpopulation of splenocytes mainly composed of B cells. Third, SpA-dependent boosted presentation of an Ag-Ab complex is further enhanced when splenocytes are enriched in cells containing SpA receptors. Fourth, the boosting effect largely diminishes when splenocytes are depleted of cells containing SpA receptors. Fifth, the boosting effect occurs only when IBP simultaneously contains a Fab and an Fc binding site. Altogether, our data suggest that soluble IBPs can bridge immune complexes to APCs containing IBP receptors, raising the possibility that during an infection process by bacteria secreting these IBPs, Ag-specific T cells may activate IBP receptor-containing B cells by a mechanism of intermolecular help, thus leading to a nonspecific immune response.

Crystal Structure of the B Subunit of Escherichia coli Heat-labile Enterotoxin Carrying Peptides with Anti-herpes Simplex Virus Type 1 Activity

Two chimeric proteins, consisting of the B subunit of Escherichia coli heat-labile enterotoxin with different peptides fused to the COOH-terminal ends, have been crystallized and their three-dimensional structure determined. The two extensions correspond to (a) a nonapeptide representing the COOH-terminal sequence of the small subunit of herpes simplex virus type 1 ribonucleotide reductase and (b) a 27-amino acid long peptide, corresponding to the COOH-terminal end of the catalytic subunit (POL) of DNA polymerase from the same virus. Both proteins crystallize in the P41212 space group with one pentameric molecule per asymmetric unit, corresponding to a solvent content of about 75%. The overall conformation of the B subunit pentamer in the two chimeric proteins, which consists of five identical polypeptide chains, is very similar to that in the native AB complex and conforms strictly to 5-fold symmetry. On the contrary, the peptide extensions are essentially disordered: in the case of the nonapeptide, only 5 and 6 amino acids were, respectively, positioned in two monomers, while in the other three only 2 residues are ordered. The extension is fully confined to the surface of the pentamer opposite to the face that interacts with the membrane and consequently it does not interfere with the ability of the B subunit to interact with membrane receptors. Moreover, the conformational flexibility of the two peptide extensions could be correlated to their propensity for proteolytic processing and consequent release of a biologically active molecule into cultured cells.

Mucosal adjuvanticity and immunogenicity of LTR72, a novel mutant of Escherichia coli heat-labile enterotoxin with partial knockout of ADP-ribosyltransferase activity.

Heat-labile Escherichia coli enterotoxin (LT) has the innate property of being a strong mucosal immunogen and adjuvant. In the attempt to reduce toxicity and maintain the useful immunological properties, several LT mutants have been produced. Some of these are promising mucosal adjuvants. However, so far, only those that were still toxic maintained full adjuvanticity. In this paper we describe a novel LT mutant with greatly reduced toxicity that maintains most of the adjuvanticity. The new mutant (LTR72), that contains a substitution Ala --> Arg in position 72 of the A subunit, showed only 0.6% of the LT enzymatic activity, was 100,000-fold less toxic than wild-type LT in Y1 cells in vitro, and was at least 20 times less effective than wild-type LT in the rabbit ileal loop assay in vivo. At a dose of 1 microg, LTR72 exhibited a mucosal adjuvanticity, similar to that observed with wild-type LT, better than that induced by the nontoxic, enzymatically inactive LTK63 mutant, and much greater than that of the recombinant B subunit. This trend was consistent for both the amounts and kinetics of the antibody induced, and priming of antigen-specific T lymphocytes. The data suggest that the innate high adjuvanticity of LT derives from the independent contribution of the nontoxic AB complex and the enzymatic activity. LTR72 optimizes the use of both properties: the enzymatic activity for which traces are enough, and the nontoxic AB complex, the effect of which is dose dependent. In fact, in dose-response experiments in mice, 20 microg of LTR72 were a stronger mucosal adjuvant than wild-type LT. This suggests that LTR72 may be an excellent candidate to be tested in clinical trials.

Monitoring the insecticidal toxins from Bacillus thuringiensis in soil with flow cytometry.

The accumulation and persistance in soil and other natural habitats of the insecticidal toxins from Bacillus thuringiensis may result in environmental hazards, such as toxicity to nontarget species and the selection of toxin-resistant target species. We describe the use of flow cytometry as a method for detecting and tracking the fate of these insecticidal toxins in soil that does not require their extraction and purification. The toxins from B. thuringiensis subspp. tenebrionis and kurstaki were bound on clay- or silt-sized particles separated from Kitchawan soil that was unamended (naturally contains predominantly kaolinite) or amended to 6% v/v with the clay minerals montmorillonite or kaolinite (as an internal control). The particle-toxin mixtures were suspended in 0.1 M phosphate buffer (pH 7) containing 3% nonfat milk powder to block nonspecific binding of antibody, resuspended in a solution of antibody to the toxin from B. thuringiensis subsp. tenebrionis, and then resuspended in a solution of anti-rabbit antibody conjugated with fluorescein isothiocyanate (FITC-Ab). Controls consisted of the particles alone and bound complexes of the particles with the toxin from B. thuringiensis subsp. kurstak. All particles that bound the toxin from B. thuringiensis subsp. tenebrionis showed a significant shift in the peak of fluorescence to the right on the x axis as compared with the nonspecific fluorescence from the control FITC-Ab complexes with particles in the absence of the toxin. There was also a slight shift in the peak to the right for some particles that bound the toxin from B. thuringiensis subsp. tenebrionis, as there is some cross-reactivity between the toxins from B. thuringiensis subspp. tenebrionis and kurstaki and the antibodies that they induce. This method is more sensitive and rapid than the dot-blot ELISA, and processing of many samples is easily accomplished.