Absence of detectable transgenes in local landraces of maize in Oaxaca, Mexico (2003-2004).

In 2000, transgenes were detected in local maize varieties (landraces) in the mountains of Oaxaca, Mexico [Quist, D. & Chapela, I. H. (2001) Nature 414, 541-543]. This region is part of the Mesoamerican center of origin for maize (Zea mays L.), and the genetic diversity that is maintained in open-pollinated landraces is recognized as an important genetic resource of great cultural value. The presence of transgenes in landraces was significant because transgenic maize has never been approved for cultivation in Mexico. Here we provide a systematic survey of the frequency of transgenes in currently grown landraces. We sampled maize seeds from 870 plants in 125 fields and 18 localities in the state of Oaxaca during 2003 and 2004. We then screened 153,746 sampled seeds for the presence of two transgene elements from the 35S promoter of the cauliflower mosaic virus and the nopaline synthase gene (nopaline synthase terminator) from Agrobacterium tumefaciens. One or both of these transgene elements are present in all transgenic commercial varieties of maize. No transgenic sequences were detected with highly sensitive PCR-based markers, appropriate positive and negative controls, and duplicate samples for DNA extraction. We conclude that transgenic maize seeds were absent or extremely rare in the sampled fields. This study provides a much-needed preliminary baseline for understanding the biological, socioeconomic, and ethical implications of the inadvertent dispersal of transgenes from the United States and elsewhere to local landraces of maize in Mexico.

gp96-peptide vaccination of mice against intracellular bacteria.

This work demonstrates that gp96 preparations isolated from cells infected with intracellular bacteria induce cytotoxic T-lymphocyte responses and confer protection. Our findings extend previous reports on the immunogenicity of gp96-associated peptides to antigens derived from intracellular bacteria. Immunization with gp96 may therefore represent a promising vaccination strategy against bacterial pathogens.

Influence of mycobacterial virulence and culture condition on gamma delta T cell activation.

Activation of human gamma delta T cells by culture supernatants of virulent and avirulent mycobacteria was examined. The stimulatory potential of mycobacteria was influenced by the type of culture media and independent from their virulence. Activation of gamma delta T cells by phagocytes infected with viable virulent Mycobacterium tuberculosis H37Rv and avirulent M. bovis BCG was comparable. We conclude that gamma delta T cell stimulation occurs in response to infection with mycobacteria independent from their virulence.

Low-molecular-weight protein ligands from Onchocerca volvulus preferentially stimulate the human gammadelta T cell Vdelta1+ subset.

Onchocerciasis is a chronic infectious disease caused by the filarial nematode Onchocerca volvulus. A minor population of human gammadelta T cells expressing Vdelta1 chains is preferentially stimulated by O. volvulus ligands in vitro. Therefore, the nature of the parasite ligand and the effector functions of Vdelta1+ T cells stimulated by O. volvulus was investigated. A 5- to 30-kDa ligand from the adult parasite lysate that is sensitive to proteinase treatment was identified. Presentation for preferential stimulation of Vdelta1+ T cells required processing. After in vitro stimulation with O. volvulus in the presence of interleukin-2, Vdelta1+ T cells produced interferon-gamma but not interleukin-4 and exhibited NK cytolytic activities. It is concluded that somatic 5- to 30-kDa protein ligands from O. volvulus stimulate Vdelta1+ T cells and that Vdelta1+ T cells play a role in immunity to O. volvulus.

Rapid determination of gamma delta T-cell stimulation by microfluorimetry.

Activated T-cell express CD25, the p55 chain of the IL-2 receptor. Here we report a reliable procedure for rapid determination of human gamma delta T cell activation by microfluorimetric measurement of CD25. Three days after initiation of culture, CD25 expression was determined. The sensitivity of this detection method was compared with [3H]thymidine incorporation at day 8. This proliferation assay allowed 3-5-fold higher dilution of the stimulatory ligand. However, monitoring of CD25 expression speeded up screening by 5 days. Therefore, for rapid screening of gamma delta T cell stimulation, e.g. for identification of activating ligands, monitoring of CD25 at day 3 is superior to [3H]thymidine measurement.

Quantification of protein in dilute and complex samples: modification of the bicinchoninic acid assay.

The colorimetric assay using bicinchoninic acid (BCA) as test reagent is useful for quantitative protein determinations due to its high sensitivity, ease, and tolerance to various contaminations present in biological samples or added during purification. For removal of interfering substances, protein precipitations have been described. Yet, obstructions became apparent with diluted and complex samples. Therefore we tested different solvents for removal of such interfering contaminants from the protein precipitate, and 1 M HCl was identified as the useful washing agent. The protocol described allows simple and accurate microdetermination in microtiter plates of proteins from complex samples.

Crossrecognition by CD8 T cell receptor alpha beta cytotoxic T lymphocytes of peptides in the self and the mycobacterial hsp60 which share intermediate sequence homology.

Immunization of C57BL/6 mice with the mycobacterial heat shock protein (hsp) 60 in immunostimulating complexes caused the in vivo activation of autoreactive major histocompatibility complex class I (H-2Db)-restricted CD8 T cell receptor (TcR) alpha/beta cells. A CD8 TcR alpha/beta clone with specificity for the mycobacterial hsp60 peptide499-508 was derived from this immunization, which, in addition, recognized syngeneic macrophages which had been stressed by interferon-gamma (IFN-gamma) stimulation. The stress-induced, self peptide could be extracted from IFN-gamma-stressed macrophages by acid elution, suggesting that the IFN-gamma-induced self peptide is derived from an endogenous protein. Based on our observation that lysis of stressed target cells by this cytotoxic T lymphocyte (CTL) clone was specifically inhibited by hsp60-specific antisense oligonucleotides, we used synthetic peptides representing amino acid (aa) sequences of the murine hsp60 for target cell sensitization and identification of the relevant self peptide. Synthetic peptides representing 9-mer to 11-mer aa sequences of the murine hsp60 with asparagine in anchor position 4 or 5 as the minimal requirement for H-2Db binding were tested in CTL assays. The overlapping murine hsp60 peptides162-170/171 were stimulatory at a concentration as low as 10-100 pM. Seven other peptides of the murine hsp60 required intermediate peptide concentrations of 10-100 nM for recognition by the CTL clone. Although the murine and mycobacterial hsp60 peptides recognized by this CTL clone showed only intermediate homology (3 identical and 3 similar aa), our data suggest that endogenous hsp60 itself is the source of self peptide(s) presented by IFN-gamma-stressed macrophages to the cross-reactive CTL clone with promiscuous specificity. This notion is consistent with the idea of hsp as a link between infection and autoimmunity.

Elongated peptides, not the predicted nonapeptide stimulate a major histocompatibility complex class I-restricted cytotoxic T lymphocyte clone with specificity for a bacterial heat shock protein.

The peptides recognized by an H-2Db-restricted CD8 cytotoxic T lymphocyte (CTL) clone which is specific for the 60-kDa mycobacterial heat shock protein (hsp) and cross-reacts with stressed host cells were characterized. None of the nonapeptides from hsp60 conforming to the H-2Db binding motif were able to sensitize target cells for lysis by this CTL clone. Sequence analysis of the stimulatory fraction from a trypsin digest of hsp60, together with synthetic peptide studies, defined a cluster of overlapping epitopes. Carboxy-terminal extension by at least one amino acid of the nonamer predicted to bind best to H-2Db was essential for CTL recognition. Two such elongated peptides, a 10-mer and a 12-mer stimulated the clone at similarly low concentrations in the 100 pM range. We assume that these two peptides comply best with the natural epitope. In contrast, the 11-mer was inactive. The stimulatory 10-mer bound to H-2Db with an efficacy similar to that of the nonapeptide corresponding to the H-2Db motif, as revealed by peptide induced major histocompatibility complex (MHC) surface expression on RMA-S cells and competitive blocking of epitope recognition by the nonamer. Binding of these carboxy-terminally extended peptides to the MHC groove can be explained by anchoring through the amino acid residue Asn in position 5 of the peptide and by intrusion of the hydrophobic carboxy-terminal Ala (10-mer) or Leu (12-mer), but not Gly (11-mer), into the hydrophobic pocket of the H-2Db cleft. Because the carboxy-terminal part is thus larger than predicted, this region of the peptide may arch up from the binding groove. We assume that recognition of steric components of the MHC/peptide complex broaden the range of epitope specificity for a single T cell receptor. This flexibility not only promotes recognition of several overlapping peptides from a single antigen, but may also increase the chance of cross-reaction with similar peptides from unrelated proteins, including autoantigens. Consistent with this latter assumption, the T cell clone cross-recognizes mycobacterial hsp60 and stressed host cells.

Beta 2-microglobulin independent presentation of exogenously added foreign peptide and endogenous self-epitope by MHC class I alpha-chain to a cross-reactive CD8+ CTL clone.

CD8+ T cells recognize antigenic peptides in the context of MHC class I molecules that encompass two distinct polypeptide chains, the MHC-encoded alpha-chain and the non-MHC-encoded beta 2-microglobulin (beta 2-m). The beta 2-m is considered essential for the stability and function of the MHC class I peptide complex and, hence, for peptide presentation to CD8+ T cells. In this study, we describe peptide presentation by macrophages from beta 2-m-deficient mice to a CD8+ CTL clone tht cross-recognizes an H-2Db-restricted peptide of the mycobacterial heat shock protein 60 (hsp60) and a self-peptide presented by IFN-gamma-stressed macrophages. Specific lysis of stressed or hsp60 peptide-pulsed beta 2-m-/- macrophages was inhibited by the nucleoprotein peptide with high affinity to H-2Db. Brefeldin A, a known inhibitor of MHC class I processing, interfered with lysis of IFN-gamma-stressed, but not of hsp60 peptide-pulsed, beta 2-m-/- macrophages. The hsp60 peptide failed to stimulate surface expression of H-2Db in beta 2-m-/- macrophages, and slightly increased MHC class I expression in the transporter mutant cell line RMA-S, as detected by cytofluorometry. We concLude that presentation of endogenously processed cytosolic epitopes and exogenously added foreign peptides by the MHC class I alpha-chain can occur independent from beta 2-m. Presumably, H-2Db peptides, but not H-2Kb peptides, have the capacity to induce and/or stabilize surface expression of a small number of MHC class I alpha-chains, and this low density is sufficient for recognition by CD8+ CTL, although it need not be detected by serologic means.

Phosphate is essential for stimulation of V gamma 9V delta 2 T lymphocytes by mycobacterial low molecular weight ligand.

T lymphocytes are divided into two subsets which express different T cell receptor heterodimers. In the peripheral blood of healthy individuals, the majority of T cells express the alpha/beta T cell receptor (> 90%) while a minority have the gamma/delta T cell receptor (< 10%). The gamma/delta T cells of adults use preferentially the V gamma 9V delta 2 chain combination. Although the stimulation requirements for gamma/delta T lymphocytes are still undetermined, it has been reported that gamma/delta T cells are not only stimulated, like alpha/beta T cells, by conventional protein antigens and superantigens, but also by unusual ligands. Mycobacteria selectively stimulate V gamma 9V delta 2 T cells, and a nonproteinacious low molecular weight fraction of 1-3 kDa has been identified as the tentative active component. Here, we confirm the nonproteinacious nature of this ligand, and show that it is comprised of unusual carbohydrate and phosphate. Importantly, cleavage of the terminal phosphate by alkaline phosphatase completely abrogates the stimulatory activity of the low molecular weight ligand for V gamma 9V delta 2 T cells. Even mycobacterial whole lysate loses its stimulatory activity, for this T cell subset, after dephosphorylation with alkaline phosphatase. These findings identify phosphocarbohydrates as a novel molecular entity with selective stimulatory activity for a defined T cell subset.

Sequence homology of the diabetes-associated autoantigen glutamate decarboxylase with coxsackie B4-2C protein and heat shock protein 60 mediates no molecular mimicry of autoantibodies.

Molecular mimicry between viral antigens and host proteins was often suggested to be involved in induction of autoimmune diseases. In type 1 diabetes where pancreatic beta cells are destroyed by autoimmune phenomena, a linear sequence homology between a major autoantigen, glutamate decarboxylase (GAD), and the 2C protein of coxsackie B4 was identified. In addition, a sequence homology between GAD and the mycobacterial heat shock protein 60 was described and the suggestions were made that molecular mimicry between GAD, coxsackievirus B4-2C protein, and/or heat shock protein 60 (hsp60) may be actively involved in an autoimmune reaction towards the pancreatic beta-cells. Our group was the first to isolate human monoclonal autoantibodies to GAD (MICA 1-6) from a patient with newly diagnosed type 1 diabetes. The MICA allowed a detailed characterization of the diabetes associated self-epitopes in GAD and represent a set of GAD autoantibodies present in sera from patients with type 1 diabetes. Using deletion mutants of GAD we demonstrated that the regions of GAD covering the homology sequences to coxsackievirus B4 and to the hsp60 were absolutely required for binding of the MICA to GAD. We now designed an antibody-based analysis to ask whether molecular mimicry between GAD and coxsackie B4-2C or hsp60 is relevant in type 1 diabetes. Since part of the MICA recognize conformational epitopes, they allow to test for conformational molecular mimicry in viruses that have been incriminated in the development of type 1 diabetes. Our data reveal no crossreactivity between the diabetes associated GAD epitopes defined by the MICA and hsp60, rubellavirus, cytomegalovirus, and coxsackie B1-B6 virus antigens. Neither coxsackie B4-specific antibodies in sera from normal individuals nor GAD-positive sera from patients with type 1 diabetes indicated a crossreactivity between coxsackie B4-2C and GAD. Although the regions in GAD homologous to coxsackie B4-2C and hsp60 represented parts of GAD indispensible for binding of diabetes associated autoantibodies they did not mediate a crossreactivity of autoantibodies between GAD and these two proteins. No evidence for molecular mimicry between GAD and a whole panel of foreign antigens was detected by autoantibodies in type 1 diabetes.

Antigen-specific T-cell responses during primary and secondary Listeria monocytogenes infection.

Although murine listeriosis is a widely used experimental model for the analysis of cell-mediated immunity, there is little information about individual T-cell antigens of Listeria monocytogenes which are recognized during primary and secondary infection. To study the antigen responses of L. monocytogenes-reactive T cells, somatic and secreted listerial proteins were separated by two-dimensional gel electrophoresis and subsequently divided into 480 liquid fractions. Antigen-specific T cells isolated from mice at different times of primary and secondary listeriosis were tested for their capacity to proliferate with distinct protein fractions. Supernatants of these cultures were screened for the production of gamma interferon, interleukin-4 (IL-4), and IL-10. Proliferation of antigen-specific T cells correlated with the production of high concentrations of gamma interferon, whereas IL-4 and IL-10 production in response to listerial protein fractions could not be detected. During both primary and secondary listeriosis, T cells recognized a multitude of somatic and secreted proteins rather than one or a few dominant antigens. Secreted proteins were recognized before somatic proteins, and T cells recognized different fractions in secreted and somatic proteins.

Hydrophobic interaction chromatography for the purification of cytolytic bacterial toxins.

The usefulness of hydrophobic interaction chromatography for the simple purification of cytolytic bacterial toxins was studied. Conditions are described for different hydrophobic interaction chromatographic media for purifying with high yields two different kinds of such haemolysins, the thiol-activated toxin listeriolysin O from Listeria monocytogenes and alpha-toxin from Staphylococcus aureus. For listeriolysin O, purification on butyl-Sepharose was followed by gel filtration chromatography. From butyl-Sepharose the recovery of 22%. Alpha-toxin was obtained by a single purification step from alkyl-Superose with 80% recovery and a specific activity of 29,000 U/mg. On sodium dodecyl sulphate polyacrylamide gel electrophoresis purified listeriolysin O and alpha-toxin showed a single band. Another thiol-activated toxin, streptolysin O from group A streptococci, showed a recovery of 38% from butyl-Sepharose. The results suggest the feasibility of using hydrophobic interaction chromatography, particularly with columns of weak hydrophobicity, for the purification of bacterial haemolysins in high yield.

Induction of arteriosclerosis in normocholesterolemic rabbits by immunization with heat shock protein 65.

Previous studies have established the presence of high numbers of activated T lymphocytes and "aberrant" expression of major histocompatibility complex class II antigens by endothelial and smooth muscle cells in human atherosclerotic lesions, implicating the involvement of a local cellular immune response. The identity of the antigen(s) eliciting this immune response, the extent of their effect, and the atherogenic stage at which they occur remain to be determined. In the present studies, 120 normocholesterolemic New Zealand White rabbits were immunized one or more times with various antigens, with or without adjuvants. The antigens and adjuvants included human or rabbit atherosclerotic lesion proteins, ovalbumin, Freund's complete and/or incomplete adjuvants, recombinant mycobacterial heat shock protein 65 (hsp65), and two hsp-free adjuvants, Ribi complete adjuvant and lipopeptide. In addition, some groups received a high-cholesterol diet. Sixteen weeks after the first immunization the animals were killed, and arteriosclerotic lesions in the intima of the aortic arch were found to have developed only in those animals immunized with antigenic preparations containing hsp, either in the form of whole mycobacteria or as purified recombinant hsp65, although their serum cholesterol levels were normal. No arteriosclerotic changes exceeding those of controls were found in the other groups, irrespective of the antigen used. Immunohistopathologic examination revealed that the lesions contained 20% T cells, 10-30% macrophages, and 10-40% smooth muscle cells. Analysis of the peripheral blood T-lymphocyte proliferative responses revealed that the occurrence of lesions was positively correlated with the presence of hsp65-reactive T cells, suggesting that hsp65 is involved in the induction of arteriosclerotic lesions. Furthermore, combined immunization with hsp-containing material and a cholesterol-rich diet provoked development of significantly more severe atherosclerosis and the appearance of characteristic foam cells. We conclude that an (auto)immune response to hsp may initiate the development of atherosclerosis and that a high blood cholesterol level is only one albeit a very important risk factor.

Secreted antigens of Mycobacterium tuberculosis: characterization with T lymphocytes from patients and contacts after two-dimensional separation.

Little is known about T cell antigens involved in immunity against Mycobacterium tuberculosis. Most model systems use in vitro culture of human T lymphocytes with bacterial lysates or secreted proteins as antigens. In this study, proteins from 3-week-old M. tuberculosis culture filtrates were separated by two-dimensional PAGE and subsequently transferred into soluble phase. The resulting 480 fractions were screened with T lymphocytes from tuberculosis patients and healthy contacts. T cells from all 9 patients and from 8 of 10 tuberculin-positive contacts preferentially responded to a cluster of acidic proteins (pI 4-5) with molecular masses of 30-100 kDa, although they also recognized a number of other fractions. In contrast, of 7 tuberculin-negative contacts, 4 were not and 3 were only weakly stimulated by this cluster region. Therefore, this distinct cluster of secreted proteins seems to comprise dominant T cell antigens of M. tuberculosis.

Heterogeneity of the repertoire of T cells of tuberculosis patients and healthy contacts to Mycobacterium tuberculosis antigens separated by high-resolution techniques.

In this report, we describe studies to examine the repertoire of freshly isolated human T lymphocytes to 400 distinct antigen fractions of Mycobacterium tuberculosis separated by a novel method involving two-dimensional gel electrophoresis. Separated antigens were probed with T cells from tuberculosis patients and purified protein derivative (PPD)-positive (PPD+) and PPD-negative (PPD-) contacts as well as normal healthy donors. T cells from all donors tested responded to separated antigens. Stimulation profiles for tuberculosis patients and PPD+ contacts were extremely heterogeneous, formally demonstrating that an enormous number of different antigens serve as targets of the cellular immune response to M. tuberculosis. Stimulation profiles for tuberculosis patients and PPD+ contacts were indistinguishable. However, stimulation profiles for tuberculosis patients and PPD+ contacts were easily distinguishable from those for PPD- contacts. Normal healthy donors showed T cell responses similar to those of either PPD+ or PPD- contacts.

T-cell responses of leprosy patients and healthy contacts toward separated protein antigens of Mycobacterium leprae.

Sonicated extracts of Mycobacterium leprae were separated by two-dimensional gel electrophoresis and electroeluted into 400 distinct soluble fractions. These fractions were probed with T lymphocytes from leprosy patients of different disease types, healthy contacts, and unexposed healthy individuals. Proliferative responses were visualized using three-dimensional stimulation profiles. T cells from many patients and contacts responded to a multitude of antigen fractions of different molecular masses and isoelectric points. T cells from unexposed individuals gave significant responses to lysates or whole organisms of M. leprae, but no or only marginal responses to separated antigen fractions. T cells of polar tuberculoid (TT) and the majority of polar lepromatous (LL) leprosy patients responded only to separated antigen fractions but not to lysates or whole organisms of M. leprae. The remaining LL patients were totally unresponsive and even failed to respond to separated M. leprae fractions. Thus, in some leprosy patients unresponsiveness to M. leprae seems to be caused by distinct components and can be broken by using separated antigen fractions; whereas in others, anergy remains. T cells of borderline tuberculoid (BT) patients, who were under chemotherapy, responded to separated antigen fractions as well as to lysates of M. leprae organisms. In contrast, BT patients who were untreated failed to react with any of the M. leprae preparations. Similarly, T cells of the majority of LL patients responding to separated fractions were under chemotherapy; whereas T cells from untreated LL patients gave no or only marginal responses to any of the M. leprae antigen preparations. These findings suggest some linkage between the degree of T-cell responsiveness and antileprosy drug treatment.

A lectin-binding, protease-resistant mycobacterial ligand specifically activates V gamma 9+ human gamma delta T cells.

Bacterial (exogeneous) superantigens have been defined as bifunctional proteinaceous molecules. They bind to class II MHC molecules of presenting cells and engage with particular TCR-V beta gene elements, thereby activating alpha beta T cells in a V beta-oriented fashion. In previous studies we have elucidated that gamma delta T cells exhibit a propensity to vigorously respond toward mycobacterial Ag. Intrigued by this finding we now analyzed whether mycobacteria express a superantigen for a subset of human gamma delta T cells definable by the selective use of TCR-V gene elements. Here we describe that a protease-resistant, low m.w. (1 to 3 kDa) component of mycobacteria selectively activates gamma delta T cells expressing TCR-V gamma 9 gene segments. Contained in mycobacterial lysates it stimulates TCR-V gamma 9-positive gamma delta T cells at a frequency of 1/6. Stimulation is critically dependent on the presence of class II MHC-positive presenting cells, the important structure being HLA-DR molecules. The fine specificity of the V gamma 9 seeking mycobacterial ligand differs from the gamma delta T cell-stimulating structures expressed by Daudi cells. In addition, the mycobacterial, V gamma 9-seeking ligand is bound selectively to lectins such as UEAI, SBA, and DBA. We conclude that mycobacteria contain a component that acts as a superantigen for human gamma delta T cells and we believe it is this property that explains the vigorous participation of gamma delta T cells in mycobacterial infections.

Tuberculosis and leprosy: attempts to identify T-cell antigens of potential value for vaccine design.

Tuberculosis and leprosy are chronic bacterial infectious diseases which represent major health problems worldwide. It is generally accepted that, on the one hand, effective vaccination strategies are required for satisfactory control of these diseases and, on the other hand, that currently available vaccination measures are insufficient for this purpose. Ideally, a subunit vaccine should be designed which is composed of one or a few protective antigens. In this brief treatise our approach towards the identification of antigens with potential value for vaccine design is described. It comprises high resolution fractionation by two-dimensional gel electrophoresis, transfer of separated fractions by electroelution and testing of separated fractions with viable T cells and accessory cells. Using this approach we find: (1) multiple antigens are recognized by T cells from leprosy and tuberculosis patients as well as healthy contacts; (2) apparently, suppressive antigens exist in leprosy; (3) an antigen cluster which is apparently indicative for immunity against M. tuberculosis is present among secreted proteins. We hope that further improvement of this methodology will help in the rational design of subunit vaccines against tuberculosis and leprosy.

Hydrophobic interaction chromatography for the purification of a mycobacterial heat shock protein of relative molecular mass 60,000.

A recombinant mycobacterial heat shock protein of relative molecular mass 60,000 was purified by hydrophobic interaction chromatography. Chromatographic media with ligands of medium hydrophobicity, such as phenyl-Sepharose, bound too strongly to be used for the purification of this heat shock protein. Butyl-Sepharose, with weak hydrophobicity, allowed binding and elution with decreasing concentrations of ammonium sulphate, but only alkyl-Superose allowed the separation of two similar proteins from the Escherichia coli clone expressing the recombinant heat shock protein (relative molecular mass 60,000) of Mycobacterium bovis BCG. The binding parameters of recombinant human heat shock proteins of relative molecular mass 60,000 and 70,000 indicate that phenyl-Sepharose also binds too strongly for the separation of these two heat shock proteins.